Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

Sadia Ahmed; Fatima Ashraf; Muhammad Tariq; Arsalan Zaidi

doi:10.1186/s13213-021-01645-5

Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

Ahmed, Sadia; Ashraf, Fatima; Tariq, Muhammad; Zaidi, Arsalan 2021-12-01 00:00:00 Purpose: Cucumber fermentation is traditionally done using lactic acid bacteria. The involvement of probiotic cultures in food fermentation guarantees enhanced organoleptic properties and protects food from spoilage. Methods: Autochthonous lactic acid bacteria were isolated from spontaneously fermented cucumber and identified to species level. Only strains adjudged as safe for human consumption were examined for their technological and functional characteristics. Strain efficiency was based on maintaining high numbers of viable cells during simulated GIT conditions and fermentation, significant antioxidant activity, EPS production, nitrite degradation, and antimicrobial ability against Gram-positive and Gram-negative foodborne pathogens. Result: Two strains, Lactiplantibacillus plantarum NPL 1258 and Pediococcus pentosaceus NPL 1264, showing a suite of promising functional and technological attributes, were selected as a mixed-species starter for carrying out a controlled lactic acid fermentations of a native cucumber variety. This consortium showed a faster lactic acid-based acidification with more viable cells, at 4% NaCl and 0.2% inulin (w/v) relative to its constituent strains when tested individually. Sensory evaluation rated the lactofermented cucumber acceptable based on texture, taste, aroma, and aftertaste. Conclusion: The results suggest that the autochthonous LAB starter cultures can shorten the fermentation cycle and reduce pathogenic organism’ population, thus improving the shelf life and quality of fermented cucumber. The development of these new industrial starters would increase the competitiveness of production and open the country’s frontiers in the fermented vegetable market. Keywords: Fermented cucumbers, Lactic acid bacteria, Functional properties, Inulin, Starter co-cultures * Correspondence: azlanzaidi@yahoo.com National Probiotic Laboratory, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Faisalabad 38000, Pakistan Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad 45650, Pakistan © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 2 of 23 Introduction commercial manufacturing, which eliminates vegetable- Raw fruits and vegetables constitute foods of high nutri- associated lactic acid bacteria if any. Very few local stud- tional and functional value with fetching health- ies have attempted looking at local LAB resources for promoting effects (Güney and Güngörmüşler 2020). Cu- potential use as starter cultures tailored for vegetable cumber (Cucumis sativus), primarily of Asian origin, has fermentation. global appeal (Mukherjee et al. 2013). Because the vege- This work aims to collect LAB isolates from spontan- table’s juicy consistency renders it vulnerable to swift rot eously LAB fermented cucumber, characterize and use and short shelf life, considerable quantities are wasted, autochthonous mixed starter strains to guarantee safety, causing economic loss (Di Cagno et al. 2008). Fermenta- functionality, and sensory properties of lacto-fermented tion offers an alternative route for prolonging its avail- cucumber. ability and transforming them into probiotic carriers handy for consumers with milk allergies and lactose in- Materials and methods tolerances (Karasu et al. 2010). Leadoff micro-composition and selection Fermented pickles are homemade products in most Isolation of LAB from lab-made spontaneously fermented parts of the world, usually obtained by spontaneous fer- (SF) cucumber mentation (Zieliński et al. 2017). However, spontaneous Fresh and tender cucumbers (organically and conven- fermentation being an uncontrolled, highly variable tionally grown) were procured from the local fruit mar- process necessitates (Sáez et al. 2018) the use of a con- ket of Islamabad and Faisalabad, respectively. trolled manufacturing process employing consortia of Cucumbers were washed and cut into (approximately 2 beneficial microbial autochthons for large-scale food × 7 cm) pieces, and every 200 g of these slices were dis- making where sensory, nutritional, and technological at- pensed into 500 mL airtight jars followed by the addition tributes could be more consistently assured (Garma- of 400 mL of the 3% (w/v) sterile brine solution. Jars sheva et al. 2019). Autochthonous strains have the edge were incubated at ambient temperature for fermentation over allochthonous strains in being more niche-specific. of cucumber for 3 weeks. Natural fermentation was Presently, no autochthonous lactic acid bacteria (LAB) allowed on its own, depending on the naturally present starter is available for vegetable fermentation worldwide microbes. to give competitive high-quality products (Sáez et al. Fermented cucumbers (20 g) were blended with sterile 2018). Therefore, finding LAB strains as autochthonous saline solution (0.85% NaCl) in a stomacher (ProBlend candidates for cucumber fermentation with dual func- Synbiosis, UK) for 2 min at high speed (400 strokes/ tion as bioprotective agents is highly prized. In contrast min), and the cell suspension was removed from the to the choices of LAB starters available for dairy, meat, stomacher bags. Cucumber cell suspension and brine and baked good fermentations, relatively few have been samples were appropriately diluted in sterile saline solu- used for vegetable fermentations (Behera et al. 2020), tion (0.85% NaCl) and plated on De Man, Rogosa, and with only a fraction of these have any purported pro- Sharpe (MRS) agar (Merck, Germany) supplemented biotic potential (Guan et al. 2020). with 0.05% L-cysteine (Oxoid, UK) to select for LAB. LAB species such as the heterofermentative Lactoba- Presumptive LAB were isolated from spontaneously fer- cillus plantarum and L. pentosus and the homofermenta- mented cucumbers under aerobic and anaerobic condi- tive Pediococcus spp. abound on the cucumber surfaces tions (Bactron-300, Shel Lab, USA) with 5% H ,5%CO 2 2, and present an opportunity to be assessed as a starter and 90% N . Plate Count Agar (PCA) (Merck, Germany), culture for controlled fermentation (Zhai et al. 2018). Oxytetra Glucose Yeast Agar Base (OGYE) (Himedia, Mixed starter cultures are decidedly better in acidifica- India), and MacConkey Agar (LAB M Limited, UK) were tion and imparting flavors than monocultures (Nilchian used for the enumeration of total aerobic microbes, yeast et al. 2016). However, their ultimate use depends on the and molds, and fecal coliform, respectively. Streptococci competition with the preexisting indigenous microbiota and Lactococci were cultured on KF Streptococcus Agar and the sensory attributes expected of the resulting KFSA (Merck, Germany) and M17 Agar (LAB M Lim- products (Gardner et al. 2001). Ensuring good probiotic ited), and Reinforced Clostridial Agar (RCM) (Oxoid, numbers in a fermented vegetable product can be UK) was used for anaerobically isolating Clostridial spp. achieved by mixing in prebiotics such as dietary fiber (Montaño et al. 2004). Aerobic microbes such as yeasts and cellulose or inulin (Güney and Güngörmüşler 2020). and molds and fecal coliform organisms were checked Historically, fermented vegetables have been a part of using aerobic culturing conditions. the diet of the Pakistani population. Despite this, there is The isolates were chosen from different media plates a lack of commercially available autochthonous starters based on distinct cell morphology, purified, and stored suitable for the fermentation of vegetables in the coun- in 20% glycerol (v/v) at − 80 °C. The well-studied com- try. Vinegar-based pickling is the preferred mode of mercial probiotic strain Lactiplantibacilllus plantarum Ahmed et al. Annals of Microbiology (2021) 71:33 Page 3 of 23 (ATCC 8014) (Huang et al. 2013), purchased from Mi- chloramphenicol; bla, for ampicillin; aadA, aadE, and crobiologics Inc., USA, was used as a reference strain. ant(6) for Streptomycin; InuA and InuB, for clindamycin; tetM, tetK, and tetL for tetracycline (Dec et al. 2017; Phenotypic and genotypic characterization Guo et al. 2017). The enzyme profiling was performed Bacterial isolates were Gram-stained, examined micro- using a commercially available kit (API-ZYM, BioMér- scopically, and tested for catalase activity (Dash et al. ieux, France) according to the manufacturer’s instruc- 2012). Identification of the isolates was made by 16S tions. A reference strain Lactiplantibacillus plantarum rRNA gene sequencing. Following the manufacturer’s in- ATCC 8014, was additionally used. structions, total bacterial DNA was isolated using a gen- omic DNA extraction kit (Thermo Scientific, Lithuania, Probiotic potential of LAB strains European Union). The quality and concentration of GIT persistence and colonization DNA were assessed using a NanoDrop spectrophotom- Resistance to gastric acid and bile was determined using eter (Thermo Scientific 2000C, Germany). The specific a published method (Jawan et al. 2019). Log-phase bac- primer sets (Gene Link, USA) 357F (CCT ACG GGA terial cultures were incubated in PBS at pH 1.5, 3.0 and GGC AGC AG) and 926R (CCG TCA ATT CMT TTR in MRSc broth with porcine bile (Sigma-Aldrich, USA) GT) were designed to amplify the V3–V5 regions of the for 3 h at 37 °C. Bacterial growth was determined by 16S rRNA gene as previously reported (Sim et al. 2012). measuring OD every 3 h for 9 h on a SpectraMax 630 nm PCR products were sequenced on a BI3730XL 96- Plus 384 microplate reader. The phenol resistance of capillary DNA analyzer by Macrogen (Korea) using the lactobacilli was quantified by inoculating log-phase bac- same primer set as above. The sequences obtained were terial cultures at an OD of 0.1 into new MRS con- 630 nm compared with the most recently released nonredundant taining 0 to 0.4% w/v phenol (Merck, Germany) and DNA sequence database at the National Center for Bio- measuring OD every 30 min for 12 h using a Spec- 630 nm technology Information (NCBI) website http://www. traMax Plus 384 microplate reader (Jawan et al. 2019). ncbi.nlm.nih.gov/BLAST. All the identified sequences For prebiotic utilization ability, three commercially avail- were deposited with NCBI, and their accession numbers able prebiotics, namely Inulin (Alfa Aesar, Germany), were obtained. Multiple sequence analysis was done with Fructooligosaccharides (FOS), and Maltodextrin (Sigma- ClustalW. Phylogenetic analyses were conducted using Aldrich, USA) were evaluated using an established the Molecular Evolutionary Genetics Analysis (MEGA) protocol (Zago et al. 2011). Dextrose (Daejung, South version 10.0 software. Evolutionary histories were in- Korea) was used as a positive control. ferred using the maximum likelihood method with the Kimura 2-parameter model for 16S rRNA sequence ana- Effect of simulated GIT digestive process on strain viability lyses (Nel et al. 2020). The strengths of the internal The protocol for in vitro digestion of bacterial cell sus- branches of the resultant trees were statistically evalu- pensions in MRS broth and acidified cucumber brine ated by bootstrap analysis with 100 bootstrap and the recipe for GIT fluids were adapted from previ- replications. ously reported work (Weiss and Jespersen 2010). Briefly, log-phase cultures of test LAB strains were individually Selection of prospective probiotic cum starter candidates resuspended in acidified brine and MRS broth to an Hemolysis and gelatinase activity was measured using a OD of 0.6. About 2.4 mL of each of these bacterial 630 nm prescribed protocol (Kaktcham et al. 2018). A Strepto- suspensions was then separately added to 1 mL of fresh TM coccus pyogenes strain (ATCC 19615 ) was used as a saliva solution and incubated for 5 min 37 °C. The simu- positive control for hemolysis (Songisepp et al. 2012) lated gastric digestion was initiated by mixing 6.4 mL of and Bacillus cereus (ATCC 1178) for gelatinase. The bio- simulated gastric juice (pH 3.0) with salivary phase cellu- genic amine production of the LAB strains was mea- lar resuspension and incubated for another 2 h at 37 °C. sured in a medium consisting of decarboxylase agar Finally, the 10 mL of these cell suspensions were then (Himedia, India) having 2% (w/v) of one of the following: mixed with simulated duodenal juice (6 mL), bile solu- precursor amino acids L-histidine, L-ornithine (Scharlau, tion (3.0 mL), and 1 M sodium bicarbonate solution (1.0 Spain), or L-tyrosine (Alfa Aesar, Germany) (Ji et al. mL) (Merck, Germany) and again incubated for a further 2013). Antibiotic susceptibility testing (AST) was per- 2 h at 37 °C. All the digestion steps were performed formed using a commercial kit (E-Test, BioMérieux, under agitated conditions at 100 rpm (heating/cooling France) according to recommendations of the European microplate shaker, VWR, USA). After simulated diges- Food Safety Authority (EFSA), and strains were classified tion, the mixture’s total cell viability was determined by as resistant or susceptible, as previously reported (EFSA plating on MRS media using a spiral plater and Q-count 2018). Strains were further screened for the presence of system (Advanced Instruments, USA). A method de- resistant genes using gene-specific primers catA, cat for scribed by Zago et al. (2011) was used to perform the Ahmed et al. Annals of Microbiology (2021) 71:33 Page 4 of 23 lysozyme resistance assay. The hydrophobic nature of Starter potential of LAB strains the tested strains was measured using bacterial adhesion The enzymes involved in anti-nutritional tannin and to hydrocarbons (BATH) protocol with xylene as solvent gallate metabolism were determined as described before (Merck, Germany) and autoaggregation according to the (Sáez et al. 2018) with some modifications. The ability of methods previously described (Kaktcham et al. 2018). LAB to deplete sodium nitrite was determined as de- scribed by Ren et al. (2014). EPS production of the iso- lates was confirmed by the method described before Host-benefiting attributes (Anandharaj et al. 2015). Total EPS (expressed as mg/L) For coaggregation assay, logarithmic phase cultures of was evaluated in each sample using glucose as standard LAB strains grown in MRS broth at 37 °C and pathogen (50–500 mg/L), and values were expressed as mean ± strains (Escherichia coli ATCC 25922, S. pyogenes ATCC standard deviation of triplicates. The proteolytic activity 19615, Staphylococcus aureus ATCC 25923, Pseudo- of LAB strains was determined using the spectrophoto- monas aeruginosa ATCC 15442, Citrobacter freundii metric assay described by Sáez et al. (2018). The results ATCC 8090, and B. cereus ATCC 11778) grown in Nu- were expressed in mmol of free amino acids (FAA) per trient broth (Oxoid, UK) also at 37 °C were used. The liter of milk by referring to a standard curve of L- pathogen strains were all purchased from Microbiologics leucine. Activities were classified as low, intermediate, Inc., USA. Coaggregation was quantified as before and high (0–1, 1–2, and > 2 mmol/L, respectively). Tol- (Kaktcham et al. 2018). erance to saline stresses was evaluated by assessing the Six food-associated pathogenic species, namely, E. coli growth of microorganisms in MRS broth supplemented (ATCC 25922), S. pyogenes (ATCC 19615), S. aureus with 2, 4, 7, or 10% (m/V) NaCl (Daejung, South Korea), (ATCC 25923), P. aeruginosa (ATCC 15442), C. freundii respectively. Growth was determined through OD 600 nm (ATCC 8090), and B. cereus (ATCC 11778), were used increase measured at intervals during 24 h of incubation to assess the antibacterial activity of the LAB strains. at 37 °C (Sáez et al. 2018). The LAB isolates were spotted onto MRS agar plates (1 μL, ~ 10 cfu/mL, ~ 5 mm diameter) and incubated Strain screening for compatibility in mixed consortia for 24 h and then overlaid with 0.8% (w/v) soft TSB agar Statistical differences among the isolates were pointed premixed with 10 cfu/mL of indicator strain. The plates out through the Principal Components Analysis (PCA) were examined after 24 h of incubation for the presence done by the method given by Kumari et al. (2016). PCA of a zone of inhibition. The zone diameter of inhibition makes it possible to distinguish between various poten- (ZDI) was measured and interpreted as strong when ZDI tial Lactobacilli strains and identify the most promising > 20 mm; 10–20 mm, intermediate; and weak when < starter culture. The relationship among the strains was 10 mm (Halder et al. 2017). determined by PCA using XLSTAT™ software. Eleven Two complementary methods (hydroxyl radical and discriminating variables (acid and bile tolerance, hydro- superoxide anion scavenging) were performed to evalu- phobicity, auto and coaggregation, antimicrobial, EPS, ate the antioxidant activity of the tested strains as re- proteolytic activity, antioxidant activity, cholesterol as- ported (Ren et al. 2014). For evaluating BSH activity, similation, and nitrite degradation) were assessed in 10 LAB strains were tested using a plate assay method (Ji potential LAB strains. PCA was based on the model of et al. 2013) on MRS agar medium supplemented with varimax rotation. 0.37 g/L CaCl (Daejung, Korea) and 0.5% (w/v) different The compatibility of selected starter strains was deter- bile salts: sodium tauroglycocholate TGC, sodium tauro- mined through agar diffusion and cross-streak assay deoxycholate TDC (Himedia, India), sodium taurochol- (Sáez et al. 2018). ate TC (Chem-Impex International, Inc., USA), and sodium deoxycholate DC (Sigma-Aldrich, USA). The Using autochthonous starters for lacto-fermentation of capacity to assimilate cholesterol was tested in MRS cucumber broth using 100 μg/mL water-soluble PEG-Cholesterol Cucumber juice medium (CJM) was prepared as de- (Sigma Aldrich, USA) (Tomaro-Duchesneau et al. 2014). scribed elsewhere (Gardner et al. 2001) with a few modi- Carbohydrate fermentation was determined with a Hi- fications. Fresh organically grown cucumbers were Carbo Kit (Himedia, India). Log phase cultures of select blended using an automatic juice extractor (Black and strains were resuspended to a turbidity of 0.5 OD Decker food factory FX1000, Turkey). The extracted 600 nm and added to individual wells containing one of the 35 juice was centrifuged (10,000 × g, 20 min, 4 °C), then sugars and incubated at 37 °C for 24 and 48 h. Amylo- filter-sterilized through a 0.22 μm filter (Millipore Cor- lytic, lipolytic, and phytase activities of potential starter poration, Bedford, MA 01730, USA) and stored at − 20 cultures were determined aerobically and anaerobically °C before use. Select L. plantarum NPL 1258 and P. pen- (Taheri et al. 2009). tosaceus NPL 1264 were grown on MRS agar plates, and Ahmed et al. Annals of Microbiology (2021) 71:33 Page 5 of 23 a colony of bacteria was transferred separately into tasting pickles and Lacto-fermented products. Sensory filter-sterilized cucumber juices. The inoculated cucum- attributes (taste, texture, crunchiness, saltiness, sharp- ber juice was incubated at 37 °C for 48 h until the inocu- ness, aroma, flavor, color/appearance, and aftertaste) lated juices were very turbid because of the growth of were evaluated using a 5-point hedonic scale (where 1 = the inoculated bacterial cells. dislike extremely and 5 = like extremely) (Güney and Fresh and tender cucumbers (organic) were procured Güngörmüşler 2020). The panelists received samples from the local fruit market of Islamabad and Faisalabad, distributed in randomly labeled transparent polypropyl- respectively. Cucumbers (approximately 2 × 7 cm in ene cups that they could try once. The evaluation was length) were washed and blanched for 15 s at 80 °C repeated twice on two separate occasions. Sensory evalu- (Reina et al. 2005) and cubed. Approximately 200 g of ation data were presented as means of the panelists’ these cubes were dispensed into 500-mL airtight jars, scores. A standard t test was used to test for the statis- followed by the addition of 400 mL of one of the follow- tical significance of the differences observed between the ing sterile brine solutions (4% w/v NaCl, pH 4): scores of the two tests. Control: no added bacterial culture Statistical analysis A: with L. plantarum culture All samples were tested twice, and each experimental B: with 0.2% w/v inulin and L. plantarum culture parameter was determined in triplicate. Results are C: with P. pentosaceus culture expressed as mean ± SD. Statistical tests were one-way D: with 0.2% w/v inulin and P. pentosaceus culture analyses of variance (ANOVA). When effects were sig- E: with L. plantarum and P. pentosaceus cultures nificant (P < 0.05), Tukey’s test was used as a post hoc F: with 0.2% w/v inulin plus L. plantarum and our P. test. All statistical analyses were done using GraphPad pentosaceus cultures Prism software (version 9 for Windows, GraphPad Soft- ware Inc, USA). For the inoculum of starter culture, the bacteria were removed from the cucumber juice medium (CJM) by Results centrifugation at 3824 × g. The cell pellet was washed Original microbiological profile of SF cucumber and twice with saline solution and centrifuged. The final cell choice of probiotic cum starter LAB pellet was resuspended into an equal volume of saline Forty-four bacterial isolates were obtained from lab- solution. The jars were inoculated with the washed cells made spontaneously fermented cucumber (Table 1) and with an initial 107 cfu/mL population and were incu- identified based on physiological, biochemical, and geno- bated at ambient temperature for fermentation. typic characteristics. Fermented cucumbers (organically grown) were enriched with Lactobacillus (46%), whereas Microbiological & biochemical analysis Enterococcus (27%) were abundantly isolated from con- The brines of the cucumber samples were analyzed dur- ventionally grown cucumbers. Five other genera were ing the period of fermentation. One milliliter of the sam- also frequently encountered in conventional-farmed fer- ple was aseptically transferred to 9 mL of sterile saline mented cucumbers, including Pediococcus, Bacillus, Leu- solution, and appropriate dilution was poured on the conostoc, Staphylococcus, and Citrobacter. MRS agar plates for LAB, and the nutrient agar was in- The 16s rDNA gene sequences of all isolates have cubated at 37 °C for 24–48 h to determine the aerobic been submitted to the Gene bank database, and the mesophilic bacteria. Yeast and molds were enumerated phylogenetic relationship between LAB was mapped on OGYE media (Oxytetra Glucose Yeast Agar Base), based on the 16S rDNA sequences from evolutionary fecal coliforms on MacConkey agar, and Streptococci distances (Fig. 1). The maximum-likelihood method and Lactococci on M17 agar (Montaño et al. 2004). based on the Kimura 2-parameter model was used with During fermentation, the pH of brine samples was 100 bootstraps in Molecular Evolutionary Genetics Ana- regularly measured using a digital pH meter (model lysis (MEGA) software, and three main clusters were HI99161, Hanna Instruments, Germany), and the lactic identified. The first group included 27 strains of L. plan- and acetic acids produced in fermentation were mea- tarum, and two strains of P. pentosaceus clustered to- sured using a commercial kit (Megazyme, USA). gether, followed by Enterococcus strains, whereas non- LAB strains clustered discretely from the LAB strains. Sensory analysis Strains of E. faecium, E. faecalis, and few strains of L. Sensory evaluations of the fermented samples were car- plantarum exhibited characteristics rendering them un- ried out at the end of the process by a panel of 20 safe for human probiotic consumption. Both strains of healthy individuals, all in the 25–35-year age bracket, E. faecalis were β hemolytic, gelatinase positive, and half male and half female, all reasonably familiar with were resistant to aminoglycosides and tetracycline. All Ahmed et al. Annals of Microbiology (2021) 71:33 Page 6 of 23 Table 1 Background details of bacterial isolates from fermented cucumber Portion of veg. Fermented organic cucumber Fermented non-organic cucumber Aerobic Anaerobic Aerobic Anaerobic Brine NPL 1277, L. plantarum ɷ NPL 425, L. plantarum ɶ NPL 1288, E. faecalis ɷ NPL 1304, E. faecium ɶ NPL 1279, L. plantarum ɷ NPL 427, L. plantarum ɶ NPL 1289, E. coli ɷ NPL 1286, L. plantarum ɶ NPL 1280, L. plantarum ɷ NPL 428, L. plantarum ɶ NPL 1290, B. amyloliquefaciens ɷ NPL 1305, L. pseudomesenteroides ɶ NPL 1273, S. epidermidis ʨ NPL 429, L. plantarum ɶ NPL 1291, P. pentosaceus ɷ NPL 1306, L. plantarum ɶ NPL 430, L. plantarum ɶ NPL 1292, E. hirae ɷ NPL 431, L. plantarum ɶ NPL 1298, E. cloacae ʉ NPL 432, L. plantarum ɶ NPL 1299, Enterobacter sp. ʉ NPL 433, L. brevis ɶ NPL 1281, B. contaminans ʨ NPL 434, L. plantarum ɶ NPL 1295, Enterococcus sp. ʛ NPL 436, L. plantarum ɶ NPL 1296, E. cloacae ʛ NPL 437, L. plantarum ɶ NPL 1297, E. cloacae ʛ NPL 1259, L. plantarum ɸ NPL 1264, P. pentosaceus ɸ Pulp NPL 1282, B. halotolerans ɷ NPL 438, L. plantarum ɶ NPL 1287, K. pneumoniae ɷ NPL 1301, E. faecium ɸ NPL 1284, L. plantarum ʉ NPL 440, L. plantarum ɶ NPL 1300,C. amalonaticus ʉ NPL 1302 , E. hirae ɸ NPL 441, L. plantarum ɶ NPL 1303, E. faecalis ɸ NPL 442, L. plantarum ɶ NPL 443, S. thermophilus ɶ NPL 1258, L. plantarum ɶ ɶ: MRS supplemented with L-cysteine ɷ: Plate Count Agar (PCA) ɸ: Reinforced Clostridial Agar (RCM) ʉ: Oxytetra Glucose Yeast Agar Base (OGYE) ʛ: KF Streptococcus agar KFSA ʨ: MacConkey agar strains of E. faecium showed resistance to penicillin and with theincreaseofincubationtime(Table 3). aminoglycosides. Three of the L. plantarum strains were Among the L. plantarum strains, NPL 1258, NPL α hemolytic, six were gelatinase positive, three were 1277, NPL 1284, and NPL 1286 exhibited the highest found to produce biogenic amines, and three strains auto-aggregation (85–100%) after 24 h of incubation. showed aminoglycosides and lincomycin resistance In contrast, other strains showed moderate auto- genes. The remaining ten L. plantarum strains and all P. aggregation (20–50%). pentosaceus were sensitive to antibiotics, did not pro- The growth of L. plantarum (NPL 1258, NPL 1259, duce biogenic amines, were non-hemolytic and gelati- NPL 1286) and P. pentosaceus (NPL 1264) was markedly nase negative, thus deemed safe for use as starter cum less at pH 1.5 than at pH 3.0 (Table 3). All strains of L. probiotic inocula (Table 2). plantarum except for NPL 1286 could well tolerate the exposure to 0.15% (w/v) porcine bile. However, the sur- Select Lactobacillus and Pediococcus strains demonstrate vival rate was found to be low for L. plantarum strains probiotic traits NPL 1286. All the strains of L. plantarum and P. pento- LAB strains exhibit colonization and GIT persistence saceus strains were also tolerant to phenol and lysozyme. potential All the strains grew strongly in the presence of inulin None of our strains was found to be strongly hydropho- (achieving 80% growth on glucose) (Fig. 2). L. plantarum bic, but three strains, NPL 1258, NPL 1279, and NPL strains NPL 1258 and NPL 1286 and P. pentosaceus 1280, belonging to L. plantarum, were moderately strain NPL 1291 utilized inulin the most (84%, 85%, and hydrophobic (< 70%). The rest exhibited low hydropho- 71%, respectively). Fructo-oligosaccharide utilization was bicity (< 36%) (Table 3). observed in NPL 1291, NPL 1284, and NPL 1258. Some Auto-aggregation results of test strains were highly of the test strains were poorly fermentative of variable. Auto-aggregation of LAB strains increased maltodextrin. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 7 of 23 Fig. 1 Molecular phylogenetic analysis. The maximum-likelihood method based on the Kimura 2-parameter model was used with 100 bootstrap replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary analyses were performed in megaX LAB strains exhibit good tolerance of simulated human The buffering effect of the vegetable matter was most digestion pronounced for L. plantarum strain NPL 1258 and P. Food matrix effectively buffered tested LAB strains pentosaceus strain NPL 1264 which registered a nonsig- against simulated digestive fluids (Fig. 3). The simulated nificant decrease in number on exposure to simulated gastric fluid being more deleterious (1–1.5 log decrease) digestive fluids, whereas the L. plantarum strains NPL than simulated duodenal fluids (0.5–1.0 log decrease). 1279 and NPL 1306 were most vulnerable to digestive Ahmed et al. Annals of Microbiology (2021) 71:33 Page 8 of 23 action irrespective of whether they are enclosed in a None of the strains isolated in this study showed amylo- vegetable matrix or not. lytic, lipolytic, or phytase activities (data not shown). Select LAB exhibit promising host-benefiting traits LAB strains display good starter aptitude The antimicrobial spectrum of all LAB strains against All selected L. plantarum strains and P. pentosaceus six pathogenic bacteria was demonstrated by the agar strains were positive for tannase and gallate decarboxyl- overlay method. Some strains of L. plantarum variably ase activities (Table 4). In this research, all strains used inhibited the growth of pathogen indicators (Table 4). could produce EPS ranging from 88.91 to 193.7 mg/L Among the L. plantarum strains NPL 1258 and NPL (Table 5). Among them, L. plantarum NPL 1258 pro- 1259 and P. pentosaceus strain NPL 1280 were most sig- duced copious amounts of EPS, 193.7 mg/L. EPS pro- nificantly antagonistic. Table 5 showed that all the duction was the least in P. pentosaceus strain NPL 1291 strains could co-aggregate with the pathogens tested, al- (88.91 mg/L). Moreover, all the L. plantarum strains dis- though the magnitude varied from strain to strain. L. played intermediate proteolytic activity (FAA 1 to 2 plantarum strains NPL 1258 and P. pentosaceus NPL mmol/L), whereas P. pentosaceus strains exhibited low 1264 exhibited the highest coaggregation with all patho- proteolytic activity (FAA < 1 mmol/L). The maximum gens tested (63–97%) following a 4-h incubation period. proteolytic activity value (1.89 ± 0.11 mmol/L) was All strains demonstrated deconjugation with sodium found for L. plantarum NPL 1258, whereas the mini- tauroglycocholate (TGC) and sodium taurocholate (TC) mum value (0.55 ± 0.02 mmol/L) was seen in P. pentosa- salts with varying substrate affinity. In general, most ceus strain NPL 1291 (Table 5). strains showed the least preference towards deconjuga- All the tested strains can reduce the nitrite concentra- tion of sodium taurodeoxycholate (TDC) compared with tion (Table 5). The highest sodium nitrite depletion rates other bile salts (Table 4). were exhibited by the L. plantarum strains NPL 1258 Cholesterol concentrations in the cell culture medium (75%), P. pentosaceus NPL 1264 (69%), followed by L. decreased after the initial solution was co-cultured with plantarum NPL 1284 (65%). However, the lowest deg- all the Lactobacillus strains (Table 5). Among the tested radation rates were observed for L. plantarum, NPL strains, NPL 1258 exhibited the highest cholesterol re- 1286 (18%). These results show that most of the tested moval rates (44%). The strains NPL 1279 and NPL 1291 strains are highly effective in depleting sodium nitrite. showed weaker rates of cholesterol removal (12% and Growth in 2% NaCl was interpreted as strong when it 19%, respectively). approximated average growth (without NaCl), which was All Lactobacillus strains exhibited antioxidative activity true for several strains (6 out of 10) (Table 3). Growth in (Table 5). The scavenging rates for hydroxyl radicals the presence of 4% NaCl was typically halved by 50%. ranged from 38 to 84%, and those for superoxide anion However, several strains of L. plantarum exhibited de- radicals ranged from 44 to 85.5%. NPL 1258 strain tectable growth at concentrations as high as 7% NaCl. In showed the highest capacity to scavenge hydroxyl radi- the present study, L. plantarum strain NPL 1259 was cals (84%), and NPL 1259 showed the highest capacity most robustly saline tolerant, withstanding 2%, 4%, and to scavenge superoxide anion radicals (85.5%). 7% NaCl by achieving 81%, 72%, and 65 % respectively L. plantarum strains NPL 1258, NPL 1284, and NPL of a typical growth profile. 1286 and P. pentosaceus strain NPL 1264 can utilize a multitude of sugar moieties such as monosaccharides, Multivariate analysis of the most suitable starter culture oligosaccharides, and some trisaccharides (raffinose), Principal component analysis was used to single out the along with some sugar alcohols (sorbitol, xylitol, and most promising starter culture for carrying out directed mannitol) and glycosides (esculin and salicin). Our Lacto fermentation of cucumber. The biplot graphs on strains are unable to metabolize disaccharides (maltose, PCA analysis are presented in Fig. 4. The first two fac- lactose, and melibiose) and deoxy sugars (fucose and tors represented 49.83% of the variability. From PCA rhamnose) (Table 6). Malonate utilization seems to be analysis, L. plantarum strains NPL 1258 and NPL 1280 absent in all L. plantarum and P. pentosaceus strains. and P. pentosaceus strain NPL 1264 were more associ- The select L. plantarum strains and P. pentosaceus ex- ated with starter culture and probiotic potential charac- hibited peptidase and esterase lipase (C8) activities. Al- teristics selected as a suitable candidate for lactic acid kaline phosphatase, esterase lipase (C4, C14), trypsin, fermentation of cucumber. These three strains were fur- and α-mannosidase and α-fucosidase were not active in ther tested for their compatibility with each other for the selected strains, nor activities of enzymes such as α- mixed culture through agar diffusion assay and cross- chymotrypsin, α-galactosidase, β-glucuronidase, N- streak assay. No inhibition halos of L. plantarum strain acetyl-β-glucosaminidase, N-acetyl-β-glucosaminidase NPL 1258 cell-free supernatants against the P. pentosa- activities deemed undesirable were recorded (Table 6). ceus NPL 1264 were observable, suggesting the absence Ahmed et al. Annals of Microbiology (2021) 71:33 Page 9 of 23 Table 2 Safety assessment of the LAB strains Strain Antibiotic susceptibility/ antibiotic resistance genes Hemolytic Gelatinase Biogenic amine code activity activity production AM/ GM/ KM/ SM/(aadA, EM/ CM/ TC/(tetM, CL/ His Orn Lys Tyr bla aac(6′)- aph(3′)- aadE, ant(6)) erm(A), (InuA, tetK, tetL) (catA, aph(2″) IIIa erm(B) InuB) cat) NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- + - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ ++ - - + NPL S/- S/- S/- S/- S/- S/- S/- S/- α -+ - + - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - + - NPL S/- S/- S/- S/- S/- S/- S/- S/- α -- - + + NPL438 S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- S/- S/- S/- S/- S/- α -- + - - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- R/+ S/- S/- R/+ S/- γ -- - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- α +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- R/+ S/- S/- S/- S/- R/+ S/- β +- - - - Ahmed et al. Annals of Microbiology (2021) 71:33 Page 10 of 23 Table 2 Safety assessment of the LAB strains (Continued) Strain Antibiotic susceptibility/ antibiotic resistance genes Hemolytic Gelatinase Biogenic amine code activity activity production AM/ GM/ KM/ SM/(aadA, EM/ CM/ TC/(tetM, CL/ His Orn Lys Tyr bla aac(6′)- aph(3′)- aadE, ant(6)) erm(A), (InuA, tetK, tetL) (catA, aph(2″) IIIa erm(B) InuB) cat) NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL R/+ S/- S/- R/+ S/- S/- S/- S/- γ -- - - - NPL S/- R/+ S/- S/- S/- S/- R/+ S/- β +- - - - NPL R/+ S/- S/- R/+ S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ - + - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - AM ampicillin, GM gentamycin, KM kanamycin, SM streptomycin, EM erythromycin, TC tetracycline, CM clindamycin, CL chloramphenicol, His histidine, Orn, ornithine, Lys lysine, Tyr tyrosine. Results are means of three independent experiments with three repetitions ± SD (n = 3). Table 3 Probiotic potential characteristics of the selected LAB strains Strain pH tolerance (%) Bile tolerance (%) Hydrophobicity Autoaggregation (%) NaCl Lysozyme Phenol code (%) tolerance resistance resistance (%) (0.4%) 1.5 3 0.15 0.30 Xylene t t t t 247 10 2 4 6 24 NPL 45.07 ± 73.24 ± 70.22 ± 38.89 ± 54 ± 0.02 20 ± 32 ± 46 ± 87 ± +++ - + + 1258 0.01 0.1 0.04 0.03 0.02 0.01 0.12 0.01 NPL 49.86 ± 62.43 ± 62.35 ± 27.41 ± 27 ± 0.01 11 ± 15 ± 18 ± 25 ± +++ - + + 1259 0.03 0.02 0.03 0.12 0.01 0.01 0.02 0.01 NPL 44.74 ± 67.01 ± 73.33 ± 38.18 ± 30 ± 0.04 13 ± 19 ± 28 ± 33 ± +++ - + + 1264 0.05 0.03 0.02 0.04 0.03 0.04 0.03 0.11 NPL 46.67 ± 73.33 ± 49.02 ± 13.73 ± 22 ± 0.0 28 ± 36 ± 50 ± 88 ± ++- - + + 1277 0.01 0.05 0.01 0.03 0.0 0.0 0.04 0.03 NPL 20.18 ± 46.49 ± 43.31 ± 19.75 ± 41 ± 0.11 34 ± 39 ± 43 ± 72 ± ++- - + + 1279 0.04 0.03 0.06 0.07 0.01 0.02 0.03 0.05 NPL 16.67 ± 25 ± 46.36 ± 23.84 ± 45 ± 0.06 10 ± 17 ± 26 ± 59 ± ++- - + + 1280 0.02 0.05 0.07 0.02 0.03 0.01 0.02 0.06 NPL 25 ± 31.25 ± 31.25 ± 13.75 ± 14 ± 0.05 27 ± 39 ± 53 ± 99 ± +++ - + + 1284 0.01 0.02 0.03 0.04 0.04 0.04 0.06 0.01 NPL 33 ± 87 ± 27.27 ± 19.39 ± 16 ± 0.02 12 ± 24 ± 37 ± 98 ± +++ - + + 1286 0.03 0.05 0.02 0.01 0.03 0.05 0.03 0.00 NPL 33.68 ± 51.58 ± 57.58 ± 34.09 ± 34 ±0.01 10 ± 22 ± 33 ± 67 ± +++ - + + 1291 0.02 0.06 0.01 0.03 0.02 0.01 0.0 0.02 NPL 30.83 ± 34.72 ± 59.64 ± 13.36 ± 31 ± 0.0 12 ± 19 ± 38 ± 65 ± ++- - + + 1306 0.03 0.03 0.02 0.02 0.01 0.02 0.02 0.01 ATCC 41.02 ± 66.31 ± 61.21 ± 25.42 ± 35 ± 0.11 17 ± 36 ± 41 ± 54 ± ++- - + + 8014 0.01 0.08 0.01 0.03 0.03 0.02 0.02 0.01 Results are means of three independent experiments with three repetitions ± SD (n = 3). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 11 of 23 Lacto-fermentation of cucumber using select starter strains Lactiplantibacilllus plantarum and Pediococcus pentosaceus driven cucumber fermentation prevents spoilage Plating count determined the microbial changes in dif- ferent groups during the fermentation. In all controlled fermented cucumber samples, LAB strains were the pre- vailing microorganisms throughout the process, and the maximum population of the inoculated strain was in treatment F (Fig. 5). The initial salt and inulin concen- trations of 4% (w/v) and 0.2% respectively in the treated sample favored rapid growth of mixed strains. LAB population was 8.3 log10 cfu/mL on the sixth day of fer- mentation which only reduced slightly by the 18th day. Whereas in other treatments, bacterial viability was sig- Fig. 2 Prebiotic utilization by LAB strains during growth. 1258: L. nificantly reduced to 7 log10 cfu/mL till the 18th day of plantarum, 1259: L. plantarum, 1264: P. pentosaceus, 1277: L. fermentation. Yeast and other pathogenic bacteria were plantarum, 1280: L. plantarum, 1284: L. plantarum, 1286: L. plantarum, observed in the control group after the sixth day. The 1291: P. pentosaceus, 1301: L. plantarum. Results are means of three number of LAB is also significantly reduced after the 6th independent experiments with three repetitions ± SD, n =3 day of fermentation. Defined lacto-fermentation of cucumber driven by the high of antimicrobial substances that could prevent using lactic acid content these strains in mixed cultures. However, the limited On the first day of fermentation, the pH value of fer- growth of L. plantarum strains NPL 1280 was observed mented samples ranged from 3.9 to 4.4. Following 18 against P. pentosaceus NPL 1264. The cross-streak assay days of fermentation, the pH of the brine solutions de- showed similar results, as no evident competition was creased from 3.5 to 3 (Fig. 5). The pH drop in all treat- noticeable at sites of co-growth in a solid medium in ments was highly significant (P < 0.05) between the combination with NPL 1258 and NPL 1264, allowing control sample and others, but there were no significant their potential use as a mixed starter (results not differences among treatments during fermentation. The shown). pH drop was sharpest in treatments E and F and then plateauing to levels like others. The decrease of pH values in A and B samples were slighter than others. Lactic acid (g/100 mL) production continually increased in all inoculated samples relative to the control. Acetic acid (g/100 mL) production was negligible in all treat- ment samples except the control sample, where it spiked at the end. Sensory acceptability of lacto-fermented cucumber Sensory evaluation of fermented cucumber samples was performed at the culmination of the process using a panel of 10 non-trained persons. For flavor, the panelists gave an overall higher rank to samples made using both candidate LAB starter strains plus inulin than all other treatments (Fig. 6). The crunchiness was best appreci- ated in products made using dual than mono starters. LAB strains invariably contribute to the aroma, texture, and flavor of fermented products. The sharp increase in Fig. 3 In vitro digestion assay of the selected Lactobacilli without (a) acidity minimized the influence of spoilage bacteria and and with (b) food matrix. 1258: L. plantarum, 1259: L. plantarum, 1264: P. pentosaceus, 1277: L. plantarum, 1280: L. plantarum, 1284: L. consistently improved the microbiological and sensory plantarum, 1286: L. plantarum, 1291: P. pentosaceus, 1301: L. quality of the fermented product than would be possible plantarum. Results are means of three independent experiments in fermentations without defined starters (Tamang and with three repetitions ± SD, n =3 Tamang 2010). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 12 of 23 Table 4 Functional properties of selected LAB strains * ɷ ɣ Strain Diameter of inhibition zone (mm) Deconjugation of bile salts Decarboxylation of phenolics code S. p S. a E. c B. c P. a C. f TGC TC TDC Tann Gall NPL 1258 +++ +++ +++ ++ ++ ++ + + + + + NPL 1259 ++ ++ ++ ++ +++ ++ + + - + + NPL 1264 +++ ++ +++ ++ +++ ++ + + - + + NPL 1277 ++ ++ +++ ++ ++ ++ - + - + + NPL 1279 +++ ++ ++ ++ ++ ++ + + - + + NPL 1280 + + +++ + + + + + - + + NPL 1284 +++ +++ +++ ++ ++ ++ + + + + + NPL 1286 ++ ++ ++ ++ ++ ++ + + - + + NPL 1291 + + ++ ++ + ++ + + - + + NPL 1306 ++ +++ ++ ++ +++ ++ + + - + + ATCC 8014 ++ ++ ++ ++ ++ ++ + + - + + S. p, Streptococcus pyogenes ATCC 19615; S. a, Staphylococcus aureus ATCC 25923, E. c, Escherichia coli ATCC 25922; B. c, Bacillus cereus ATCC 11778; P. a. Pseudomonas aeruginosa ATCC 15442; C. f. Citrobacter freundii ATCC 8090 * (+) weak (< 10 mm), (++) intermediate (10–20 mm), (+++) strong (< 20 mm), (−) no zone TGC, tauroglycocholate; TC, sodium taurocholate; TDC, sodium taurodeoxycholate; DC, sodium deoxycholate (+) present, (-) absent. Results are means of three independent experiments with three repetitions ± SD (n =3) Table 5 Functional properties of selected LAB strains Strain Antioxidant activity Cholesterol Nitrite Proteolytic EPS Coaggregation (%) code assimilation degradation activity (mg/L) Hydroxyl Superoxide S. a S. p E. c B. c P. a (%) (%) (mmol/L) radical (%) anions (%) NPL 84 ± 0.22 64.65 ± 0.17 44 ± 0.12 75 ±0.01 1.89 ± 0.11 85.5 ± 77 ± 85 ± 66 ± 52 ± 52 ± 1258 0.01 0.11 0.05 0.02 0.01 0.01 NPL 48 ± 0.12 85.5 ± 0.22 38 ± 0.02 59 ± 0.12 1.73 ± 0.0 74.9 ± 31 ± 43 ± 87 ± 41 ± 41 ± 1259 0.12 0.03 0.08 0.04 0.03 0.03 NPL 73 ± 0.14 64.9 ± 0.32 33 ± 0.11 69 ± 0.05 0.76 ± 0.0 92.1 ± 97 ± 66 ± 53 ± 63 ± 63 ± 1264 0.15 0.06 0.13 0.04 0.04 0.04 NPL 52 ± 0.31 46.15 ± 0.45 22 ± 0.21 36 ± 0.03 1.61 ± 0.16 86.8 ± 34 ± 82 ± 62 ± 47 ± 47 ± 1277 0.17 0.13 0.16 0.03 0.5 0.5 NPL 60 ± 0.11 76.85 ± 0.27 12 ± 0.12 55 ± 0.17 1.38 ± 0.09 88.6 ± 74 ± 44 ± 36 ± 54 ± 54 ± 1279 0.21 0.16 0.04 0.11 0.11 0.11 NPL 56 ± 0.16 68.65 ± 0.22 29 ± 0.05 65 ± 0.02 1.41 ± 0.05 76.1 ± 87 ± 76 ± 55 ± 48 ± 48 ± 1280 0.33 0.25 0.02 0.03 0.21 0.21 NPL 74 ± 0.15 76.31 ± 0.13 31 ± 0.02 56 ± 0.14 1.60 ± 0.02 85.2 ± 29 ± 22 ± 48 ± 63 ± 63 ± 1284 0.12 0.15 0.05 0.02 0.04 0.04 NPL 66 ± 0.06 84.18 ± 0.17 23 ± 0.16 18 ± 0.11 1.67 ± 0.11 71.9 ± 43 ± 52 ± 58 ± 33 ± 33 ± 1286 0.11 0.01 0.11 0.01 0.04 0.04 NPL 53 ± 0.11 64.98 ± 0.19 19 ± 0.41 58 ± 0.04 0.55 ± 0.02 88.91 ± 63 ± 73 ± 61 ± 16 ± 16 ± 1291 0.02 0.00 0.05 0.01 0.2 0.2 NPL 38 ± 0.09 44.34 ± 0.32 20 ± 0.01 34 ± 0.01 1.52 ± 0.03 91 ± 75.3 ± 58 ± 49 ± 82 ± 64 ± 1306 0.04 0.02 0.21 0.12 0.01 0.01 ATCC 44 ± 0.03 62.34 ± 0.02 25 ± 0.04 56 ± 0.02 0.92 ± 0.01 83 ± 66.78 ± 67 ± 56 ± 71 ± 39 ± 8014 0.01 0.2 0.11 0.11 0.01 0.02 S. p, Streptococcus pyogenes ATCC 19615; S. a, Staphylococcus aureus ATCC 25923, E. c, Escherichia coli ATCC 25922, B. c Bacillus cereus ATCC 11778. P. a Pseudomonas aeruginosa ATCC 15442. Results are means of three independent experiments with three repetitions ± SD (n = 3). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 13 of 23 Table 6 Enzyme characterization and carbohydrate utilization of selected LAB NPL NPL NPL NPL NPL NPL NPL NPL NPL NPL ATCC 1258 1259 1264 1277 1279 1280 1284 1286 1291 1306 8014 Sr. Carbohydrate no. fermentation 1 Lactose + + + + + + + + + + + 2 Xylose + + + + + + + + + + + 3 Maltose + + - + + + + + - + + 4 Fructose + - + + - + + + + + + 5 Dextrose + - + + - + + - + + + 6 Galactose + - + + - + + - + + + 7 Raffinose + - + + - + + + + - - 8 Trehalose + + + + + + + + + + + 9 Melibiose - - - - - - - - - - - 10 Sucrose + - + + - + + + + + + 11 L-Arabinose + + + + + + + + + + + 12 Mannose + - + + - + + + + + + 13 Inulin + + + + + + + + + + + 14 Sodium gluconate - - - - - - - - - - - 15 Glycerol - - - - - + + - - - - 16 Salicin + + + + + + + + + - + 17 Dulcitol - - - - - - + - - - - 18 Inositol - - - - - - - - - - - 19 Sorbitol + + + + + + + + + + + 20 Mannitol + + + + + + + + + + + 21 Adonitol - - - - - - + - - - + 22 Arabitol - - - - - - + - - + + 23 Erythritol - - - - - - - - - - - 24 α-Methyl-D glucoside - - - - - - + - - - - 25 Rhamnose - - - - - - + - - - - 26 Cellobiose + + + + + + + + + + + 27 Melezitose - + - - - - + - - - - 28 α-methyl-D mannoside + + - - - - + - - - - 29 Xylitol + + - - - - + - - - - 30 ONPG + + - - - - + - - - - 31 Esculin hydrolysis + + + + + + + + + + + 32 D-Arabinose + + + + + + + + + + + 33 Citrate utilization - + - - - + + + - - + 34 Malonate utilization - + - - - - - - - - - 35 Sorbose - - - - - - - - - - - Sr. Enzyme activity no. 1 Alkaline phosphatase 0 0 0 0 0 0 0 0 0 0 0 2 Esterase (C 4) 0 0 0 0 0 0 0 0 0 0 0 3 Esterase lipase (C 8) 5 5 10 5 10 10 10 5 10 5 5 4 Lipase (C 14) 0 0 0 0 0 0 0 0 0 0 0 5 Leucine arylamidase > 40 20 20 20 > 40 > 40 > 40 20 10 10 > 40 Ahmed et al. Annals of Microbiology (2021) 71:33 Page 14 of 23 Table 6 Enzyme characterization and carbohydrate utilization of selected LAB (Continued) NPL NPL NPL NPL NPL NPL NPL NPL NPL NPL ATCC 1258 1259 1264 1277 1279 1280 1284 1286 1291 1306 8014 6 Valine arylamidase 20 20 20 20 20 20 20 20 5 5 10 7 Cystine arylamidase > 40 > 40 20 20 20 20 20 20 20 20 20 8 Trypsin 0 0 0 0 0 0 0 0 0 0 0 9 α-chymotrypsin 0 0 0 0 0 0 0 0 0 0 0 10 Acid phosphatase 5 5 5 5 5 5 5 5 5 5 10 11 Naphthol-AS-BI- 5 555 555 555 20 phosphohydrolase 12 α-galactosidase 0 0 0 0 0 0 0 0 0 0 0 13 ß-galactosidase 20 20 > 40 20 20 > 40 20 20 20 20 20 14 ß-glucuronidase 0 0 0 0 0 0 0 0 0 0 0 15 α-glucosidase 0 0 0 0 0 0 0 0 0 0 0 16 ß-glucosidase 20 10 20 10 5 5 5 10 5 10 20 17 N-acetyl-ß-glucosaminidase 0 0 0 0 0 0 0 0 0 0 0 18 α-mannosidase 0 0 0 0 0 0 0 0 0 0 0 19 α-fucosidase 0 0 0 0 0 0 0 0 0 0 0 Enzyme activities were assessed through API-ZYM galleries that were expressed in terms of color intensity, which ranged from 0 (no activity), 5–10 (low), 30 (moderate), and ≥ 40 nmol (strong) of substrate hydrolyzed following 4 h incubation at 37 °C. Color intensity was judged with reference to an API ZYM color chart provided by the manufacturer. ‘+’ shows a positive reaction; ‘−’ shows a negative reaction Fig. 4 PCA biplot projecting the probiotic potential starter culture variables and strains Ahmed et al. Annals of Microbiology (2021) 71:33 Page 15 of 23 Fig. 5 Microbiological and physicochemical analysis of lacto-fermented cucumber during fermentation period. a Changes in viable cell number of LAB in different samples, b pH, c LA (Lactic acid), and d AA (Acetic acid) during fermentation. Control: without bacterial inocula with 4% (w/v) NaCl, pH 4; A: L. plantarum NPL 1258 with 4% (w/v) NaCl, pH 4; B: L. plantarum NPL 1258 with 4% (w/v) NaCl, 0.2% inulin, pH 4; C: P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; D: P. pentosaceus NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4; E: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; F: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4. Results are means of three independent experiments with three repetitions ± SD, n =3 Discussion Fermented cucumber is a popular culinary choice in many Asian countries (Behera et al. 2020). Fermented vegetables containing LAB are being increasingly researched because of their benefits to vegetarians and individuals with dairy intolerances (Di Cagno et al. 2013). Many vegetables with good nutritional content but otherwise poor organoleptics can be rendered flavor- ful and desirable for consumption when fermented by LAB species (Alan et al. 2018). Autochthonous LAB abounds spontaneously fermentation vegetables, an es- sentially stochastic event buffeted by numerous environ- mental parameters (Xu et al. 2018). A slew of potential autochthonous starter cultures must be examined and the right fit identified to standardize and control the Fig. 6 Sensory analysis of lacto-fermented cucumber after fermentation period. Control: without bacterial inocula with 4% (w/ process, especially in large-scale cucumber fermentations v) NaCl, pH 4; A: L. plantarum NPL 1258 with 4% (w/v) NaCl, pH 4; B: (Jampaphaeng et al. 2018). L. plantarum NPL 1258 with 4% (w/v) NaCl, 0.2% inulin, pH 4; C: P. 2 3 Generally, LAB constitute a small part (10 –10 cfu/g) pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; D: P. pentosaceus of the full spectrum of autochthonous microbiota of raw NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4; E: L. plantarum NPL vegetables (Kothari et al. 2020). Lactobacillus spp., Leu- 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; F: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) conostoc spp., and Pediococcus spp. are the leading LAB NaCl, 0.2% inulin, pH 4. Results are means of three independent predominating on the cucumber surface and are respon- experiments with three repetitions ± SD, n =3 sible for fermenting it (Behera et al. 2020). Finding Ahmed et al. Annals of Microbiology (2021) 71:33 Page 16 of 23 plentiful L. plantarum is expected since it is the pre- (Benítez-Cabello et al. 2019) and global regulatory guide- dominant Lactobacillus associated with SF cucumber be- lines emphatic stance on excluding probiotic candidates cause of its penchant for thriving in high acidity and with even a smidgen of hemolytic behavior (Food et al. salinity (Behera et al. 2018). 2006). Enzyme profiling is also helpful for selecting A multitude of LAB spp. such as L. brevis, S. thermo- strains to be used in food, as their enzymatic activities philus, E. faecium, P. pentosaceus, and Leuconostoc may affect the quality of the product (Colombo et al. mesenteroides in association with the fermented vege- 2018). table matter as seen here is in line with the notion where In recent years, it has been posited that food bacteria a broad vegetal microbial diversity could be attributed to may act as reservoirs of antibiotic resistance genes, a slew of agro-technological factors such as cultivars, which might be transferred to gut commensals or patho- geography, seasons, and aspects of the fermentation gens (Hummel et al. 2007). However, Lactobacillus, es- process like the quality of the base material used and the pecially those of fermented food origin, are unlikely to equipment surfaces (Khalil et al. 2018). Leuconostoc contribute to the AMR (Antimicrobial Resistance) prob- mesenteroides is an infrequent primary fermenter of cu- lem in humans (Ma et al. 2017). Nevertheless, the Euro- cumber whose numbers are eventually superseded by L. pean Food Safety Authority (EFSA) considers its plantarum, which finishes the process (Fusco et al. assessment a primary criterion for according “qualified 2017). A greater abundance of Enterobacteriaceae was presumption of safety” (QPS) status (Zielińska et al. observed on conventionally farmed vegetables than or- 2015). Intrinsic resistance to antibiotics such as amino- ganically farmed via culturing technique (Leff and Fierer glycosides and vancomycin in Pediococcus (Shukla and 2013). This effect could be attributed to several factors: Goyal 2014) and Lactobacillus species such as L. plan- growing location, fertilizer use, pesticide use, other agri- tarum are intrinsic, non-transferable, and not sufficiently cultural practices, and shipping and handling procedures concerning to prevent their use for food fermentation (Beuchat et al. 2006). Enterococcal species as E. hirae, E. (Abriouel et al. 2015; EFSA 2018). Therefore, all strains faecium, and E. cloacae, although naturally present on carrying antibiotic-resistant genes were adjudged as un- cucumber, are pinned down during the LAB-driven pri- safe and precluded from further study. A high incidence mary fermentation phase, only rebounding when the pH of antibiotic resistance in Lactobacillus species has been starts to rise. Since E. cloacae tends to spoil fermenting attributed to insufficient checks and balances for the use cucumbers, its presence is considered undesirable and disposal of antibiotics in developing societies (Ma (Franco and Pérez-Díaz 2013). et al. 2017). Before assaying for probiotic functionality in candidate The probiotic effectiveness of these innocuous candi- strains, their safety status was determined in vitro as rec- date starters was gauged by testing their tolerance to hu- ommended by FAO (Food et al. 2006) since probiotic man gut physiological parameters such as pH, and bile starter cultures are consumed live in large dosages tolerance must be examined in vitro (Anandharaj et al. through the fermented product; therefore, their harm- 2015). Such an approach is faster, cheaper, reproducible, lessness to public health must be ensured (Chokesajja- and offers more choice of controlled conditions disen- watee et al. 2020). LAB spp. generally have a good safety cumbered with many ethical restrictions associated with record for human consumption; however, there are some in vivo experimentation (Calvo-Lerma et al. 2019). exceptions involving L. plantarum (Cannon et al. 2005) Robust acid tolerance in a potential probiotic starter and the enterococcal species E. faecium and E. faecalis candidate, besides empowering it to survive the human (Sanchez Valenzuela et al. 2013; Strateva et al. 2016). host’s GIT milieu, also prolongs its survivability in highly LAB that are amino biogenic during spontaneous lactic acidic fermented foods (Michalak et al. 2020). Tolerating acid fermentation can jeopardize the quality and safety extreme acidity as seen in L. plantarum strains NPL of fermented foods (Alan et al. 2018), thus are ill-suited 1258, NPL 1259, and NPL 1280 is likely due to their for use as starters and adjuncts (Behera et al. 2020; Beli- adaptiveness to the pickle habitat (Adebayo-tayo and cová et al. 2013). Both candidate starters and probiotics Onilude 2008) from where they have been isolated in must incontrovertibly demonstrate an absence of harm- this study. Withstanding bile exposure is vital for an ful enzymatic activities, such as β-glucosidase and β- ingested probiotic to survive in the small intestine (Ana- glucuronidase, known to cause detrimental effects in the ndharaj et al. 2015). Tolerating 0.15–0.3% concentration colon (Zielińska et al. 2015). Testing for virulence attri- of bile salts is a sufficient threshold for any probiotic butes such as hemolysins, typically associated with path- taken orally (Alp and Aslim 2010). The extent to which ogens, was also deemed essential because of a past Lactobacilli can withstand bile is crucial since its levels precedent of its occurrence, albeit a sporadic one in in the gut are not static, fluctuating from 1.5 to 2% (w/v) some Lactobacillus species (Domingos-Lopes et al. in the first hour of digestion and eventually plateauing 2017), including L. plantarum strains of vegetable origin out at 0.3% (w/v) (Bao et al. 2010). LAB strains of Ahmed et al. Annals of Microbiology (2021) 71:33 Page 17 of 23 vegetable origins are generally less bile resistant (Chiu autoaggregative strength (Lee et al. 2014). However, the et al. 2008), contrasting with our findings where some L. extent to which cell hydrophobicity correlates with self plantarum strains were significantly bile resistant. The and coaggregation can vary in LAB spp. (Li et al. 2015). adaptation mechanism to bile salts is multifactorial. It is Coaggregation is advantageous because it allows a pro- attributed to changes in the bacterium’s ability to fer- biotic strain to produce antimicrobial substances in ment carbohydrates, exopolysaccharides production, the proximity to several pathogen cells, effectively eliminat- balance of proteins and fatty acids in its cell membrane, ing them from the GIT (Tuo et al. 2013). and the ability to firmly adhere to human mucus (Ali Among the technological properties much sought in et al. 2020). LAB are EPS production, salt tolerance, and antimicro- Any prospective probiotics added to a food matrix bial metabolites. The ability of LAB to produce EPS is a should tolerate food processing and storage conditions common trait of LAB starters as it helps to improve the and the entire swathe of gastrointestinal transit following colonization of probiotic bacteria in the gastrointestinal ingestion right up to their intestinal site of action (Cam- tract and protect against the baleful effects of harmful pos et al. 2019). A high titer of probiotic bacteria in food resident bacteria (Kumar et al. 2017). Our finding of products at the point of consumption does not guaran- copiously EPS-producing L. plantarum strains from fer- tee the same numbers in the gut because stomach acid- mented cucumber has some precedent (Jiang et al. ity and intestinal bile can dramatically reduce their 2016). viability (da Cruz Rodrigues et al. 2019). Estimating the The antimicrobial activity of LAB may improve the ravages of a GIT transit can be best done by simulating quality of fermented foods by eliminating spoilage and digestive processes in vitro (Campos et al. 2019), starting pathogenic bacteria (Michalak et al. 2020). Our results from the mouth to the ileum, and factoring in the effects align with previous findings where L. plantarum strains of the food matrix, enzymes, and peristalsis (Neffe-Sko- inhibited gram-positive bacteria such as S. aureus, S. cińska et al. 2018). Aside from its simplicity, low cost, pyogenes, and B. cereus, and Gram-negative C. freundii, and high throughput format, the clincher in this method E. coli, and P. aeruginosa (Gheziel et al. 2019; Jiang et al. is sequential exposure to acid and bile, a more authentic 2016). Indeed, antagonistic activity against E. coli is a depiction of actual physiological events. Our findings of relevant screening criterion because of the frequent L. plantarum and Pediococcus strains capable of with- presence of coliforms in cucumber pickle brine (Lu et al. standing GIT transit without any significant deleterious- 2013). Other food quality indicator microorganisms are ness support previous reports (Barbosa et al. 2015; also essential to test due to their high load on cucumber Gheziel et al. 2019). L. plantarum derived from SF cu- surfaces. Pediococcus species such as P. pentosaceus are cumber are likely to be intrinsically strong acid-tolerant inhibitory of pernicious and ubiquitous human patho- because the pickled vegetables mimic harsh gastrointes- gens such as L. monocytogenes, which has been challen- tinal conditions with pH values reaching three or lower ging to control with standard industrial approaches (Cao et al. 2019). (Huang et al. 2009). One of the caveats of a good probiotic is that the Since cucumber fermentation occurs in brine with 5– strain must also endure the action of toxic metabolites 7% NaCl (Di Cagno et al. 2008), candidate LAB starter (primarily phenols) produced during the digestion strains ought to be halotolerant, as is the case here process (Jawan et al. 2019), which is also observed here. (Rodriguez-Palacios et al. 2017). Some strains of L. plan- The selected L. plantarum and P. pentosaceus strains tarum were exceptionally tolerant of salt, which may be harbored a significant ability to metabolize food pheno- due to the accumulation of osmo and cryoprotective sol- lics via active tannase and gallate decarboxylases, de- utes such as betaine and carnitine (Yao et al. 2020). grading tannin and gallic acids. Both are anti-nutritional Halotolerance is not a staple characteristic of the species factors (ANF) that inactivate digestive enzymes, as much variability is observed (Ziadi et al. 2019). insolubilize proteins, and affect the utilization of vita- Carbohydrate utilization by lactic acid bacteria (LAB) mins and minerals (Sáez et al. 2018). Microbial metabo- defines the extent of cucumber fermentations, their lization of nitrite during cucumber fermentation is also quality, and long-term stability. Aside from glucose and advantageous since dietary nitrite is linked to gastro- fructose, alternate energy sources such as trehalose, cel- intestinal cancers (Behera et al. 2020), and its control is lobiose, and xylose, available in cucumber fermentations. important from a food safety standpoint (Ren et al. These compounds remained present in cucumber fer- 2014). mentations even after glucose and fructose were con- LAB cells that strongly aggregate and whose surfaces sumed. The removal of these alternate energy sources by are hydrophobic can adhere well to intestinal epithelia starter cultures during the most active period of the bio- and occlude gut luminal pathogens (Abbasiliasi et al. conversion prevents the proliferation of spoilage- 2017). BATH values are usually a proxy for associated microbes such as L. buchneri (Ucar et al. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 18 of 23 2020a; Ucar et al. 2020b). The strains of L. plantarum digestibility (Rizzello et al. 2016). A weakly proteolytic and P. pentosaceus used in this study can use trehalose behavior of LAB species found in this study could be and cellobiose and hamper the growth and metabolic ac- tied to their vegetal origins that are poor in protein con- tivity of spoilage-associated microbes. Our L. plantarum tent (Sáez et al. 2018). and P. pentosaceus strains can utilize plant-based carbo- Bile salt hydrolase (BSH) activity is essential for bac- hydrates such as mannitol and inulin (Gustaw et al. teria to thrive in the intestine and for the human host 2018; Lee et al. 2014), which helps to promote the who can benefit from its cholesterol-lowering effect growth of Lactobacillus. The raffinose-degrading ability (Jones et al. 2013). BSH hydrolyzes conjugated glyco- of our strains is an appealing feature because of their as- deoxycholic and taurodeoxycholic acid into glyco- and sociation with flatulence and GI disturbances in humans tauro-bile acids, respectively (Anandharaj et al. 2015), (Arunraj et al. 2020). Malonate utilization seems absent thereby protecting gut microbiota and probiotic bacteria in all L. plantarum and P. pentosaceus strains associated from their toxicity (Shukla and Goyal 2014). Typically, with bloating during cucumber fermentation (Bintsis LAB with vegetal origins where bile salts are absent do 2018). Our strains’ diverse metabolic profile helps them not possess bile salt hydrolase activity (Zielińska et al. endure well in various non-dairy food matrices and the 2015) which is at odds with our finding. Nevertheless, human intestine (Gupta and Bajaj 2018). both L. plantarum and P. pentosaceus have been shown An extracellular enzyme produced by the candidate to have BSHs active against tauroconjugates of bile salts probiotics in the food matrix may improve the organo- (Lee et al. 2014). This study supports previous conten- leptic properties of food and enhance human digestion tions where LAB with active bile salt hydrolase has been (Gupta and Bajaj 2018). Enzymatic profiles of LAB that shown to lower cholesterol levels, removing secondary lack proteinases but have strong peptidase and esterase bile salts and cholesterol from the human body (Peres lipase activities are associated with imparting typical and et al. 2014). The extent to which they are successful is desirable flavors to fermented vegetable products (Abba- highly strain dependent (Zhang et al. 2014). siliasi et al. 2017; Goswami et al. 2017); therefore, the Inulin, fructooligosaccharides, and maltodextrins are presence of these traits in our strains confirms their es- well-established prebiotics that remain undigested in the sential role in flavor development of fermented cucum- upper gastrointestinal tract and are only fermented by ber. Acid phosphatase and phosphohydrolase allow the Lactobacillus in the colon (Choudhary et al. 2019). In- probiotic strains to metabolize diverse substrates of the corporating them into a probiotic carrying food matrix GIT (Shokryazdan et al. 2017). The absence of harmful should have a proliferative effect on probiotic LAB, enzymes, α-glucosidases or β-glucuronidase, implicated which is the case here. LAB species have cell-associated in carcinogenesis (Yeo et al. 2016), and chymotrypsin glycosidases and fructofuranosidases for hydrolysis of and N-acetyl-B-glucosaminidase that are linked with these substrates and using the resulting monomers as an GIT dysfunction (Delgado et al. 2007) renders strains energy source (Perrin et al. 2001). Choosing the right safer for human consumption. prebiotic is deemed essential and must be empirically Strains with copious EPS production with significant determined for any novel synbiotic to succeed. We chose halotolerance and anti-pathogen capabilities were also inulin instead of FOS as the prebiotic part of the synbio- assessed for withstanding oxidative stress. The antioxida- tic for lacto-fermentation of cucumber because the tive mechanisms protect colonizing LAB from attack by former was more readily fermentable and improved free radicals while benefiting the human host by mitigat- colonization and persistence of L. plantarum (Brajdes ing cardiovascular diseases, diabetes, and ulcers of the and Vizireanu 2013). The FOS, on the other hand, is GI tract (Ren et al. 2014). It was found that some strains more suitable as prebiotic to bifidobacterial instead of of L. plantarum registered more antioxidant capacity Lactobacillus spp. in synbiotic combinations (Schrezen- than the reference strain. Vegetable matrices are typic- meir and de Vrese 2001). ally characterized as high antioxidant environments When considering starters tailored for cucumber fer- (Verni et al. 2017). An aspect that has not gone un- mentation, the selection of strains from its natural noticed since fermented vegetables’ consumption was microbiota allows for by-passing the adaptation chal- found to correlate with a lower COVID-19 pandemic lenges that allochthonous cultures could face, thus facili- mortality rate (Fonseca et al. 2020). tating an improved nutritional, functional, and The proteolytic activity of LAB enhances the organo- technological profile of fermented cucumber (Verni leptic profile of fermented foods through the release of et al. 2017). L. plantarum and Pediococcus spp. are well- free amino acids and their derivatives (Karasu et al. recognized starter cultures giving various fermented 2010; Verni et al. 2017). In addition, they are also in- vegetable products (Behera et al. 2018). Although strains volved in the degradation of allergenic and anti- could be selected based on their technological features, nutritional proteins and the increased protein multiple attributes are more helpful in making foods that Ahmed et al. Annals of Microbiology (2021) 71:33 Page 19 of 23 have better sensory properties. A principal component acid through its exclusive homofermentative pathway analysis (PCA) can facilitate screening out the most suit- (Güney and Güngörmüşler 2020). The production of able strain for fermenting vegetables (Sáez et al. 2018). acetic acid was less than the lactic acid in all inoculated Mixed species inoculation of brined cucumbers with P. cucumber samples. The decreased concentrations of lac- pentosaceus and L. plantarum has the potential advan- tic acid accompanied by increased acetic acid concentra- tage of an early, rapid initial growth and moderate acid tions in the control fermented cucumber sample could production by the former species and a higher final acid- be due to the degradation of lactic acid into acetic acid ity resulting from the lower pH tolerance of the latter by spoilage-associated microorganisms, especially L. (Tamang and Tamang 2010). buchneri (Johanningsmeier and McFeeters 2013). The typical industry standard of a minimum of 10 Sensory analysis is an indispensable tool to determine cfu/g of probiotics in a product stems from clinical in- consumer acceptability (Cuffia et al. 2018). Irrespective 8 9 vestigations where a minimum of 10 –10 cfu/g of pro- of a product’s health claims, the customer will likely re- biotic was deemed necessary for health benefits (da Cruz ject it if the sensory profile is poor (Karimi et al. 2012). et al. 2009). Maintenance of appropriate numbers of We used an affective sensory method using a 5-point he- probiotics during the storage of fermented vegetables is donic scale popular in the industry and academic re- quite challenging due to the low pH of brine, nutrient search (Greifova 2007). The concept is serially monadic depletion, and the accumulation of lactic acid (Valero- that does not allow for retasting or contextual reference Cases and Frutos 2017). The supplementation of a nat- to capture consumer purchase behavior more accurately ural plant origin prebiotic such as inulin can stimulate (Wichchukit and O'Mahony 2015). Sensory analysis is a beneficial bacteria’s growth and metabolic activity in try- human-centric propriocentric view of a food product. ing conditions (Nilchian et al. 2016). It can also protect Taste and pleasure are among the most meaningful pre- the cells from refrigeration-associated cell damage, dictors of food choice (Brunsø et al. 2002). Lacto- mainly through physical immobilization of the cells in fermented cucumbers with mixed starter cultures were inulin macroaggregates (Bedani et al. 2013). Before fer- preferable to monoculture fermented cucumber con- mentation, the addition of prebiotics improves the shelf cerning taste and pleasure. This difference might be be- life viability of probiotic bacteria mixed in with a food cause mixed strain cultures are relatively less affected by product (Szydłowska and Kołożyn-Krajewska 2019). The vicissitudes of handling, storage, and applications and pro-proliferative and protective effect of inulin in L. contribute more to desirable flavors while cutting down plantarum seems strain-dependent judging from reports on unpleasant ones (Holzapfel 2002). Bitterness and over in its favor (Valero-Cases and Frutos 2015) and disfavor sourness are negatively hedonistic (Greifova 2007), and (Nazzaro et al. 2012). mar acceptance of control spontaneously fermented cu- Starters bring about a rapid decrease of pH, which cumbers (Drewnowski and Gomez-Carneros 2000; Ver- helps to reduce the risk of spoilage at the beginning of heul et al. 2013). fermentation (Nilchian et al. 2016). The ability to acidify rapidly is desirable for any would-be LAB starter of Conclusion vegetable fermentation (Wakil et al. 2014). The ability to The present study highlights the capacity of two LAB acidify a medium is typical of many LAB species because strains, autochthonous to SF cucumber, as potential pro- of organic acids, mainly lactic acid (Greifova 2007). L. biotic cum starter culture candidates. The results indi- plantarum directed acidification of the samples has been cate that inoculated selected starters manifested in vitro applied for food preservation (Muthusamy et al. 2020). several desirable, beneficial probiotic attributes such as Optimizing brine concentration empirically as done here antioxidant, BSH activity, cholesterol assimilation, and is critical for a desirable and appropriately fermented cu- antibiotic susceptibility. In addition, the inoculated cumber (Nilchian et al. 2016). Higher concentrations of starters remained significantly viable during fermenta- brine promote spoilage because of lowered pH (Bautista- tion and contributed to the aroma and flavor of the fer- Gallego et al. 2010). mented cucumber. They prevented putrescence caused Lactic acid and acetic acid are the primary metabolic by spoilage bacteria and enhanced the sensorial aspects end products of carbohydrate fermentation during cu- of the fermented product. A high inoculum of L. plan- cumber fermentation by LAB, which lowers the food tarum and P. pentosaceus strains (NPL 1258 and NPL pH, imbuing it with desirable organoleptic properties 1259) was found to control the fermented cucumber’s and eliminating pathogens, ensuring safety and stability quality effectively. Strain probioticity, no doubt a good of the final product (Adesulu-Dahunsi et al. 2018). A sig- and valuable attribute, nonetheless does not obviate the nificant increase in lactic acid production during anaer- need to examine the fermentation kinetics and physio- obic fermentation of cucumber is associated with the logical benefits further before they could be recom- capability of L. plantarum for making substantial lactic mended for large-scale commercial application. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 20 of 23 Acknowledgments Anandharaj M, Sivasankari B, Santhanakaruppu R, Manimaran M, Rani RP, Not applicable Sivakumar S (2015) Determining the probiotic potential of cholesterol- reducing Lactobacillus and Weissella strains isolated from gherkins (fermented cucumber) and south Indian fermented koozh. Res Microbiol 166(5):428–439. Data availability https://doi.org/10.1016/j.resmic.2015.03.002 All data generated or analyzed during this study are included in this Arunraj R, Skori L, Kumar A, Hickerson NM, Shoma N, Samuel MA (2020) Spatial published article. regulation of alpha-galactosidase activity and its influence on raffinose family oligosaccharides during seed maturation and germination in Cicer arietinum. Authors’ contributions Plant Signal Behav 15(8):1709707. https://doi.org/10.1080/15592324.2019.1 The contributions of SA and AZ are equal. SA contributed to the investigation, data analysis, writing the original draft. FA contributed to the Bao Y, Zhang Y, Zhang Y, Liu Y, Wang S, Dong X, Wang Y, Zhang H (2010) investigation. MT did the validation, resources. AZ did the conceptualization, Screening of potential probiotic properties of Lactobacillus fermentum experimental design, writing, reviewing, editing, visualization, supervision, isolated from traditional dairy products. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2021
ISSN: 1590-4261
eISSN: 1869-2044
DOI: 10.1186/s13213-021-01645-5
Publisher site: See Article on Publisher Site

Abstract

Purpose: Cucumber fermentation is traditionally done using lactic acid bacteria. The involvement of probiotic cultures in food fermentation guarantees enhanced organoleptic properties and protects food from spoilage. Methods: Autochthonous lactic acid bacteria were isolated from spontaneously fermented cucumber and identified to species level. Only strains adjudged as safe for human consumption were examined for their technological and functional characteristics. Strain efficiency was based on maintaining high numbers of viable cells during simulated GIT conditions and fermentation, significant antioxidant activity, EPS production, nitrite degradation, and antimicrobial ability against Gram-positive and Gram-negative foodborne pathogens. Result: Two strains, Lactiplantibacillus plantarum NPL 1258 and Pediococcus pentosaceus NPL 1264, showing a suite of promising functional and technological attributes, were selected as a mixed-species starter for carrying out a controlled lactic acid fermentations of a native cucumber variety. This consortium showed a faster lactic acid-based acidification with more viable cells, at 4% NaCl and 0.2% inulin (w/v) relative to its constituent strains when tested individually. Sensory evaluation rated the lactofermented cucumber acceptable based on texture, taste, aroma, and aftertaste. Conclusion: The results suggest that the autochthonous LAB starter cultures can shorten the fermentation cycle and reduce pathogenic organism’ population, thus improving the shelf life and quality of fermented cucumber. The development of these new industrial starters would increase the competitiveness of production and open the country’s frontiers in the fermented vegetable market. Keywords: Fermented cucumbers, Lactic acid bacteria, Functional properties, Inulin, Starter co-cultures * Correspondence: azlanzaidi@yahoo.com National Probiotic Laboratory, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Faisalabad 38000, Pakistan Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad 45650, Pakistan © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 2 of 23 Introduction commercial manufacturing, which eliminates vegetable- Raw fruits and vegetables constitute foods of high nutri- associated lactic acid bacteria if any. Very few local stud- tional and functional value with fetching health- ies have attempted looking at local LAB resources for promoting effects (Güney and Güngörmüşler 2020). Cu- potential use as starter cultures tailored for vegetable cumber (Cucumis sativus), primarily of Asian origin, has fermentation. global appeal (Mukherjee et al. 2013). Because the vege- This work aims to collect LAB isolates from spontan- table’s juicy consistency renders it vulnerable to swift rot eously LAB fermented cucumber, characterize and use and short shelf life, considerable quantities are wasted, autochthonous mixed starter strains to guarantee safety, causing economic loss (Di Cagno et al. 2008). Fermenta- functionality, and sensory properties of lacto-fermented tion offers an alternative route for prolonging its avail- cucumber. ability and transforming them into probiotic carriers handy for consumers with milk allergies and lactose in- Materials and methods tolerances (Karasu et al. 2010). Leadoff micro-composition and selection Fermented pickles are homemade products in most Isolation of LAB from lab-made spontaneously fermented parts of the world, usually obtained by spontaneous fer- (SF) cucumber mentation (Zieliński et al. 2017). However, spontaneous Fresh and tender cucumbers (organically and conven- fermentation being an uncontrolled, highly variable tionally grown) were procured from the local fruit mar- process necessitates (Sáez et al. 2018) the use of a con- ket of Islamabad and Faisalabad, respectively. trolled manufacturing process employing consortia of Cucumbers were washed and cut into (approximately 2 beneficial microbial autochthons for large-scale food × 7 cm) pieces, and every 200 g of these slices were dis- making where sensory, nutritional, and technological at- pensed into 500 mL airtight jars followed by the addition tributes could be more consistently assured (Garma- of 400 mL of the 3% (w/v) sterile brine solution. Jars sheva et al. 2019). Autochthonous strains have the edge were incubated at ambient temperature for fermentation over allochthonous strains in being more niche-specific. of cucumber for 3 weeks. Natural fermentation was Presently, no autochthonous lactic acid bacteria (LAB) allowed on its own, depending on the naturally present starter is available for vegetable fermentation worldwide microbes. to give competitive high-quality products (Sáez et al. Fermented cucumbers (20 g) were blended with sterile 2018). Therefore, finding LAB strains as autochthonous saline solution (0.85% NaCl) in a stomacher (ProBlend candidates for cucumber fermentation with dual func- Synbiosis, UK) for 2 min at high speed (400 strokes/ tion as bioprotective agents is highly prized. In contrast min), and the cell suspension was removed from the to the choices of LAB starters available for dairy, meat, stomacher bags. Cucumber cell suspension and brine and baked good fermentations, relatively few have been samples were appropriately diluted in sterile saline solu- used for vegetable fermentations (Behera et al. 2020), tion (0.85% NaCl) and plated on De Man, Rogosa, and with only a fraction of these have any purported pro- Sharpe (MRS) agar (Merck, Germany) supplemented biotic potential (Guan et al. 2020). with 0.05% L-cysteine (Oxoid, UK) to select for LAB. LAB species such as the heterofermentative Lactoba- Presumptive LAB were isolated from spontaneously fer- cillus plantarum and L. pentosus and the homofermenta- mented cucumbers under aerobic and anaerobic condi- tive Pediococcus spp. abound on the cucumber surfaces tions (Bactron-300, Shel Lab, USA) with 5% H ,5%CO 2 2, and present an opportunity to be assessed as a starter and 90% N . Plate Count Agar (PCA) (Merck, Germany), culture for controlled fermentation (Zhai et al. 2018). Oxytetra Glucose Yeast Agar Base (OGYE) (Himedia, Mixed starter cultures are decidedly better in acidifica- India), and MacConkey Agar (LAB M Limited, UK) were tion and imparting flavors than monocultures (Nilchian used for the enumeration of total aerobic microbes, yeast et al. 2016). However, their ultimate use depends on the and molds, and fecal coliform, respectively. Streptococci competition with the preexisting indigenous microbiota and Lactococci were cultured on KF Streptococcus Agar and the sensory attributes expected of the resulting KFSA (Merck, Germany) and M17 Agar (LAB M Lim- products (Gardner et al. 2001). Ensuring good probiotic ited), and Reinforced Clostridial Agar (RCM) (Oxoid, numbers in a fermented vegetable product can be UK) was used for anaerobically isolating Clostridial spp. achieved by mixing in prebiotics such as dietary fiber (Montaño et al. 2004). Aerobic microbes such as yeasts and cellulose or inulin (Güney and Güngörmüşler 2020). and molds and fecal coliform organisms were checked Historically, fermented vegetables have been a part of using aerobic culturing conditions. the diet of the Pakistani population. Despite this, there is The isolates were chosen from different media plates a lack of commercially available autochthonous starters based on distinct cell morphology, purified, and stored suitable for the fermentation of vegetables in the coun- in 20% glycerol (v/v) at − 80 °C. The well-studied com- try. Vinegar-based pickling is the preferred mode of mercial probiotic strain Lactiplantibacilllus plantarum Ahmed et al. Annals of Microbiology (2021) 71:33 Page 3 of 23 (ATCC 8014) (Huang et al. 2013), purchased from Mi- chloramphenicol; bla, for ampicillin; aadA, aadE, and crobiologics Inc., USA, was used as a reference strain. ant(6) for Streptomycin; InuA and InuB, for clindamycin; tetM, tetK, and tetL for tetracycline (Dec et al. 2017; Phenotypic and genotypic characterization Guo et al. 2017). The enzyme profiling was performed Bacterial isolates were Gram-stained, examined micro- using a commercially available kit (API-ZYM, BioMér- scopically, and tested for catalase activity (Dash et al. ieux, France) according to the manufacturer’s instruc- 2012). Identification of the isolates was made by 16S tions. A reference strain Lactiplantibacillus plantarum rRNA gene sequencing. Following the manufacturer’s in- ATCC 8014, was additionally used. structions, total bacterial DNA was isolated using a gen- omic DNA extraction kit (Thermo Scientific, Lithuania, Probiotic potential of LAB strains European Union). The quality and concentration of GIT persistence and colonization DNA were assessed using a NanoDrop spectrophotom- Resistance to gastric acid and bile was determined using eter (Thermo Scientific 2000C, Germany). The specific a published method (Jawan et al. 2019). Log-phase bac- primer sets (Gene Link, USA) 357F (CCT ACG GGA terial cultures were incubated in PBS at pH 1.5, 3.0 and GGC AGC AG) and 926R (CCG TCA ATT CMT TTR in MRSc broth with porcine bile (Sigma-Aldrich, USA) GT) were designed to amplify the V3–V5 regions of the for 3 h at 37 °C. Bacterial growth was determined by 16S rRNA gene as previously reported (Sim et al. 2012). measuring OD every 3 h for 9 h on a SpectraMax 630 nm PCR products were sequenced on a BI3730XL 96- Plus 384 microplate reader. The phenol resistance of capillary DNA analyzer by Macrogen (Korea) using the lactobacilli was quantified by inoculating log-phase bac- same primer set as above. The sequences obtained were terial cultures at an OD of 0.1 into new MRS con- 630 nm compared with the most recently released nonredundant taining 0 to 0.4% w/v phenol (Merck, Germany) and DNA sequence database at the National Center for Bio- measuring OD every 30 min for 12 h using a Spec- 630 nm technology Information (NCBI) website http://www. traMax Plus 384 microplate reader (Jawan et al. 2019). ncbi.nlm.nih.gov/BLAST. All the identified sequences For prebiotic utilization ability, three commercially avail- were deposited with NCBI, and their accession numbers able prebiotics, namely Inulin (Alfa Aesar, Germany), were obtained. Multiple sequence analysis was done with Fructooligosaccharides (FOS), and Maltodextrin (Sigma- ClustalW. Phylogenetic analyses were conducted using Aldrich, USA) were evaluated using an established the Molecular Evolutionary Genetics Analysis (MEGA) protocol (Zago et al. 2011). Dextrose (Daejung, South version 10.0 software. Evolutionary histories were in- Korea) was used as a positive control. ferred using the maximum likelihood method with the Kimura 2-parameter model for 16S rRNA sequence ana- Effect of simulated GIT digestive process on strain viability lyses (Nel et al. 2020). The strengths of the internal The protocol for in vitro digestion of bacterial cell sus- branches of the resultant trees were statistically evalu- pensions in MRS broth and acidified cucumber brine ated by bootstrap analysis with 100 bootstrap and the recipe for GIT fluids were adapted from previ- replications. ously reported work (Weiss and Jespersen 2010). Briefly, log-phase cultures of test LAB strains were individually Selection of prospective probiotic cum starter candidates resuspended in acidified brine and MRS broth to an Hemolysis and gelatinase activity was measured using a OD of 0.6. About 2.4 mL of each of these bacterial 630 nm prescribed protocol (Kaktcham et al. 2018). A Strepto- suspensions was then separately added to 1 mL of fresh TM coccus pyogenes strain (ATCC 19615 ) was used as a saliva solution and incubated for 5 min 37 °C. The simu- positive control for hemolysis (Songisepp et al. 2012) lated gastric digestion was initiated by mixing 6.4 mL of and Bacillus cereus (ATCC 1178) for gelatinase. The bio- simulated gastric juice (pH 3.0) with salivary phase cellu- genic amine production of the LAB strains was mea- lar resuspension and incubated for another 2 h at 37 °C. sured in a medium consisting of decarboxylase agar Finally, the 10 mL of these cell suspensions were then (Himedia, India) having 2% (w/v) of one of the following: mixed with simulated duodenal juice (6 mL), bile solu- precursor amino acids L-histidine, L-ornithine (Scharlau, tion (3.0 mL), and 1 M sodium bicarbonate solution (1.0 Spain), or L-tyrosine (Alfa Aesar, Germany) (Ji et al. mL) (Merck, Germany) and again incubated for a further 2013). Antibiotic susceptibility testing (AST) was per- 2 h at 37 °C. All the digestion steps were performed formed using a commercial kit (E-Test, BioMérieux, under agitated conditions at 100 rpm (heating/cooling France) according to recommendations of the European microplate shaker, VWR, USA). After simulated diges- Food Safety Authority (EFSA), and strains were classified tion, the mixture’s total cell viability was determined by as resistant or susceptible, as previously reported (EFSA plating on MRS media using a spiral plater and Q-count 2018). Strains were further screened for the presence of system (Advanced Instruments, USA). A method de- resistant genes using gene-specific primers catA, cat for scribed by Zago et al. (2011) was used to perform the Ahmed et al. Annals of Microbiology (2021) 71:33 Page 4 of 23 lysozyme resistance assay. The hydrophobic nature of Starter potential of LAB strains the tested strains was measured using bacterial adhesion The enzymes involved in anti-nutritional tannin and to hydrocarbons (BATH) protocol with xylene as solvent gallate metabolism were determined as described before (Merck, Germany) and autoaggregation according to the (Sáez et al. 2018) with some modifications. The ability of methods previously described (Kaktcham et al. 2018). LAB to deplete sodium nitrite was determined as de- scribed by Ren et al. (2014). EPS production of the iso- lates was confirmed by the method described before Host-benefiting attributes (Anandharaj et al. 2015). Total EPS (expressed as mg/L) For coaggregation assay, logarithmic phase cultures of was evaluated in each sample using glucose as standard LAB strains grown in MRS broth at 37 °C and pathogen (50–500 mg/L), and values were expressed as mean ± strains (Escherichia coli ATCC 25922, S. pyogenes ATCC standard deviation of triplicates. The proteolytic activity 19615, Staphylococcus aureus ATCC 25923, Pseudo- of LAB strains was determined using the spectrophoto- monas aeruginosa ATCC 15442, Citrobacter freundii metric assay described by Sáez et al. (2018). The results ATCC 8090, and B. cereus ATCC 11778) grown in Nu- were expressed in mmol of free amino acids (FAA) per trient broth (Oxoid, UK) also at 37 °C were used. The liter of milk by referring to a standard curve of L- pathogen strains were all purchased from Microbiologics leucine. Activities were classified as low, intermediate, Inc., USA. Coaggregation was quantified as before and high (0–1, 1–2, and > 2 mmol/L, respectively). Tol- (Kaktcham et al. 2018). erance to saline stresses was evaluated by assessing the Six food-associated pathogenic species, namely, E. coli growth of microorganisms in MRS broth supplemented (ATCC 25922), S. pyogenes (ATCC 19615), S. aureus with 2, 4, 7, or 10% (m/V) NaCl (Daejung, South Korea), (ATCC 25923), P. aeruginosa (ATCC 15442), C. freundii respectively. Growth was determined through OD 600 nm (ATCC 8090), and B. cereus (ATCC 11778), were used increase measured at intervals during 24 h of incubation to assess the antibacterial activity of the LAB strains. at 37 °C (Sáez et al. 2018). The LAB isolates were spotted onto MRS agar plates (1 μL, ~ 10 cfu/mL, ~ 5 mm diameter) and incubated Strain screening for compatibility in mixed consortia for 24 h and then overlaid with 0.8% (w/v) soft TSB agar Statistical differences among the isolates were pointed premixed with 10 cfu/mL of indicator strain. The plates out through the Principal Components Analysis (PCA) were examined after 24 h of incubation for the presence done by the method given by Kumari et al. (2016). PCA of a zone of inhibition. The zone diameter of inhibition makes it possible to distinguish between various poten- (ZDI) was measured and interpreted as strong when ZDI tial Lactobacilli strains and identify the most promising > 20 mm; 10–20 mm, intermediate; and weak when < starter culture. The relationship among the strains was 10 mm (Halder et al. 2017). determined by PCA using XLSTAT™ software. Eleven Two complementary methods (hydroxyl radical and discriminating variables (acid and bile tolerance, hydro- superoxide anion scavenging) were performed to evalu- phobicity, auto and coaggregation, antimicrobial, EPS, ate the antioxidant activity of the tested strains as re- proteolytic activity, antioxidant activity, cholesterol as- ported (Ren et al. 2014). For evaluating BSH activity, similation, and nitrite degradation) were assessed in 10 LAB strains were tested using a plate assay method (Ji potential LAB strains. PCA was based on the model of et al. 2013) on MRS agar medium supplemented with varimax rotation. 0.37 g/L CaCl (Daejung, Korea) and 0.5% (w/v) different The compatibility of selected starter strains was deter- bile salts: sodium tauroglycocholate TGC, sodium tauro- mined through agar diffusion and cross-streak assay deoxycholate TDC (Himedia, India), sodium taurochol- (Sáez et al. 2018). ate TC (Chem-Impex International, Inc., USA), and sodium deoxycholate DC (Sigma-Aldrich, USA). The Using autochthonous starters for lacto-fermentation of capacity to assimilate cholesterol was tested in MRS cucumber broth using 100 μg/mL water-soluble PEG-Cholesterol Cucumber juice medium (CJM) was prepared as de- (Sigma Aldrich, USA) (Tomaro-Duchesneau et al. 2014). scribed elsewhere (Gardner et al. 2001) with a few modi- Carbohydrate fermentation was determined with a Hi- fications. Fresh organically grown cucumbers were Carbo Kit (Himedia, India). Log phase cultures of select blended using an automatic juice extractor (Black and strains were resuspended to a turbidity of 0.5 OD Decker food factory FX1000, Turkey). The extracted 600 nm and added to individual wells containing one of the 35 juice was centrifuged (10,000 × g, 20 min, 4 °C), then sugars and incubated at 37 °C for 24 and 48 h. Amylo- filter-sterilized through a 0.22 μm filter (Millipore Cor- lytic, lipolytic, and phytase activities of potential starter poration, Bedford, MA 01730, USA) and stored at − 20 cultures were determined aerobically and anaerobically °C before use. Select L. plantarum NPL 1258 and P. pen- (Taheri et al. 2009). tosaceus NPL 1264 were grown on MRS agar plates, and Ahmed et al. Annals of Microbiology (2021) 71:33 Page 5 of 23 a colony of bacteria was transferred separately into tasting pickles and Lacto-fermented products. Sensory filter-sterilized cucumber juices. The inoculated cucum- attributes (taste, texture, crunchiness, saltiness, sharp- ber juice was incubated at 37 °C for 48 h until the inocu- ness, aroma, flavor, color/appearance, and aftertaste) lated juices were very turbid because of the growth of were evaluated using a 5-point hedonic scale (where 1 = the inoculated bacterial cells. dislike extremely and 5 = like extremely) (Güney and Fresh and tender cucumbers (organic) were procured Güngörmüşler 2020). The panelists received samples from the local fruit market of Islamabad and Faisalabad, distributed in randomly labeled transparent polypropyl- respectively. Cucumbers (approximately 2 × 7 cm in ene cups that they could try once. The evaluation was length) were washed and blanched for 15 s at 80 °C repeated twice on two separate occasions. Sensory evalu- (Reina et al. 2005) and cubed. Approximately 200 g of ation data were presented as means of the panelists’ these cubes were dispensed into 500-mL airtight jars, scores. A standard t test was used to test for the statis- followed by the addition of 400 mL of one of the follow- tical significance of the differences observed between the ing sterile brine solutions (4% w/v NaCl, pH 4): scores of the two tests. Control: no added bacterial culture Statistical analysis A: with L. plantarum culture All samples were tested twice, and each experimental B: with 0.2% w/v inulin and L. plantarum culture parameter was determined in triplicate. Results are C: with P. pentosaceus culture expressed as mean ± SD. Statistical tests were one-way D: with 0.2% w/v inulin and P. pentosaceus culture analyses of variance (ANOVA). When effects were sig- E: with L. plantarum and P. pentosaceus cultures nificant (P < 0.05), Tukey’s test was used as a post hoc F: with 0.2% w/v inulin plus L. plantarum and our P. test. All statistical analyses were done using GraphPad pentosaceus cultures Prism software (version 9 for Windows, GraphPad Soft- ware Inc, USA). For the inoculum of starter culture, the bacteria were removed from the cucumber juice medium (CJM) by Results centrifugation at 3824 × g. The cell pellet was washed Original microbiological profile of SF cucumber and twice with saline solution and centrifuged. The final cell choice of probiotic cum starter LAB pellet was resuspended into an equal volume of saline Forty-four bacterial isolates were obtained from lab- solution. The jars were inoculated with the washed cells made spontaneously fermented cucumber (Table 1) and with an initial 107 cfu/mL population and were incu- identified based on physiological, biochemical, and geno- bated at ambient temperature for fermentation. typic characteristics. Fermented cucumbers (organically grown) were enriched with Lactobacillus (46%), whereas Microbiological & biochemical analysis Enterococcus (27%) were abundantly isolated from con- The brines of the cucumber samples were analyzed dur- ventionally grown cucumbers. Five other genera were ing the period of fermentation. One milliliter of the sam- also frequently encountered in conventional-farmed fer- ple was aseptically transferred to 9 mL of sterile saline mented cucumbers, including Pediococcus, Bacillus, Leu- solution, and appropriate dilution was poured on the conostoc, Staphylococcus, and Citrobacter. MRS agar plates for LAB, and the nutrient agar was in- The 16s rDNA gene sequences of all isolates have cubated at 37 °C for 24–48 h to determine the aerobic been submitted to the Gene bank database, and the mesophilic bacteria. Yeast and molds were enumerated phylogenetic relationship between LAB was mapped on OGYE media (Oxytetra Glucose Yeast Agar Base), based on the 16S rDNA sequences from evolutionary fecal coliforms on MacConkey agar, and Streptococci distances (Fig. 1). The maximum-likelihood method and Lactococci on M17 agar (Montaño et al. 2004). based on the Kimura 2-parameter model was used with During fermentation, the pH of brine samples was 100 bootstraps in Molecular Evolutionary Genetics Ana- regularly measured using a digital pH meter (model lysis (MEGA) software, and three main clusters were HI99161, Hanna Instruments, Germany), and the lactic identified. The first group included 27 strains of L. plan- and acetic acids produced in fermentation were mea- tarum, and two strains of P. pentosaceus clustered to- sured using a commercial kit (Megazyme, USA). gether, followed by Enterococcus strains, whereas non- LAB strains clustered discretely from the LAB strains. Sensory analysis Strains of E. faecium, E. faecalis, and few strains of L. Sensory evaluations of the fermented samples were car- plantarum exhibited characteristics rendering them un- ried out at the end of the process by a panel of 20 safe for human probiotic consumption. Both strains of healthy individuals, all in the 25–35-year age bracket, E. faecalis were β hemolytic, gelatinase positive, and half male and half female, all reasonably familiar with were resistant to aminoglycosides and tetracycline. All Ahmed et al. Annals of Microbiology (2021) 71:33 Page 6 of 23 Table 1 Background details of bacterial isolates from fermented cucumber Portion of veg. Fermented organic cucumber Fermented non-organic cucumber Aerobic Anaerobic Aerobic Anaerobic Brine NPL 1277, L. plantarum ɷ NPL 425, L. plantarum ɶ NPL 1288, E. faecalis ɷ NPL 1304, E. faecium ɶ NPL 1279, L. plantarum ɷ NPL 427, L. plantarum ɶ NPL 1289, E. coli ɷ NPL 1286, L. plantarum ɶ NPL 1280, L. plantarum ɷ NPL 428, L. plantarum ɶ NPL 1290, B. amyloliquefaciens ɷ NPL 1305, L. pseudomesenteroides ɶ NPL 1273, S. epidermidis ʨ NPL 429, L. plantarum ɶ NPL 1291, P. pentosaceus ɷ NPL 1306, L. plantarum ɶ NPL 430, L. plantarum ɶ NPL 1292, E. hirae ɷ NPL 431, L. plantarum ɶ NPL 1298, E. cloacae ʉ NPL 432, L. plantarum ɶ NPL 1299, Enterobacter sp. ʉ NPL 433, L. brevis ɶ NPL 1281, B. contaminans ʨ NPL 434, L. plantarum ɶ NPL 1295, Enterococcus sp. ʛ NPL 436, L. plantarum ɶ NPL 1296, E. cloacae ʛ NPL 437, L. plantarum ɶ NPL 1297, E. cloacae ʛ NPL 1259, L. plantarum ɸ NPL 1264, P. pentosaceus ɸ Pulp NPL 1282, B. halotolerans ɷ NPL 438, L. plantarum ɶ NPL 1287, K. pneumoniae ɷ NPL 1301, E. faecium ɸ NPL 1284, L. plantarum ʉ NPL 440, L. plantarum ɶ NPL 1300,C. amalonaticus ʉ NPL 1302 , E. hirae ɸ NPL 441, L. plantarum ɶ NPL 1303, E. faecalis ɸ NPL 442, L. plantarum ɶ NPL 443, S. thermophilus ɶ NPL 1258, L. plantarum ɶ ɶ: MRS supplemented with L-cysteine ɷ: Plate Count Agar (PCA) ɸ: Reinforced Clostridial Agar (RCM) ʉ: Oxytetra Glucose Yeast Agar Base (OGYE) ʛ: KF Streptococcus agar KFSA ʨ: MacConkey agar strains of E. faecium showed resistance to penicillin and with theincreaseofincubationtime(Table 3). aminoglycosides. Three of the L. plantarum strains were Among the L. plantarum strains, NPL 1258, NPL α hemolytic, six were gelatinase positive, three were 1277, NPL 1284, and NPL 1286 exhibited the highest found to produce biogenic amines, and three strains auto-aggregation (85–100%) after 24 h of incubation. showed aminoglycosides and lincomycin resistance In contrast, other strains showed moderate auto- genes. The remaining ten L. plantarum strains and all P. aggregation (20–50%). pentosaceus were sensitive to antibiotics, did not pro- The growth of L. plantarum (NPL 1258, NPL 1259, duce biogenic amines, were non-hemolytic and gelati- NPL 1286) and P. pentosaceus (NPL 1264) was markedly nase negative, thus deemed safe for use as starter cum less at pH 1.5 than at pH 3.0 (Table 3). All strains of L. probiotic inocula (Table 2). plantarum except for NPL 1286 could well tolerate the exposure to 0.15% (w/v) porcine bile. However, the sur- Select Lactobacillus and Pediococcus strains demonstrate vival rate was found to be low for L. plantarum strains probiotic traits NPL 1286. All the strains of L. plantarum and P. pento- LAB strains exhibit colonization and GIT persistence saceus strains were also tolerant to phenol and lysozyme. potential All the strains grew strongly in the presence of inulin None of our strains was found to be strongly hydropho- (achieving 80% growth on glucose) (Fig. 2). L. plantarum bic, but three strains, NPL 1258, NPL 1279, and NPL strains NPL 1258 and NPL 1286 and P. pentosaceus 1280, belonging to L. plantarum, were moderately strain NPL 1291 utilized inulin the most (84%, 85%, and hydrophobic (< 70%). The rest exhibited low hydropho- 71%, respectively). Fructo-oligosaccharide utilization was bicity (< 36%) (Table 3). observed in NPL 1291, NPL 1284, and NPL 1258. Some Auto-aggregation results of test strains were highly of the test strains were poorly fermentative of variable. Auto-aggregation of LAB strains increased maltodextrin. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 7 of 23 Fig. 1 Molecular phylogenetic analysis. The maximum-likelihood method based on the Kimura 2-parameter model was used with 100 bootstrap replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Evolutionary analyses were performed in megaX LAB strains exhibit good tolerance of simulated human The buffering effect of the vegetable matter was most digestion pronounced for L. plantarum strain NPL 1258 and P. Food matrix effectively buffered tested LAB strains pentosaceus strain NPL 1264 which registered a nonsig- against simulated digestive fluids (Fig. 3). The simulated nificant decrease in number on exposure to simulated gastric fluid being more deleterious (1–1.5 log decrease) digestive fluids, whereas the L. plantarum strains NPL than simulated duodenal fluids (0.5–1.0 log decrease). 1279 and NPL 1306 were most vulnerable to digestive Ahmed et al. Annals of Microbiology (2021) 71:33 Page 8 of 23 action irrespective of whether they are enclosed in a None of the strains isolated in this study showed amylo- vegetable matrix or not. lytic, lipolytic, or phytase activities (data not shown). Select LAB exhibit promising host-benefiting traits LAB strains display good starter aptitude The antimicrobial spectrum of all LAB strains against All selected L. plantarum strains and P. pentosaceus six pathogenic bacteria was demonstrated by the agar strains were positive for tannase and gallate decarboxyl- overlay method. Some strains of L. plantarum variably ase activities (Table 4). In this research, all strains used inhibited the growth of pathogen indicators (Table 4). could produce EPS ranging from 88.91 to 193.7 mg/L Among the L. plantarum strains NPL 1258 and NPL (Table 5). Among them, L. plantarum NPL 1258 pro- 1259 and P. pentosaceus strain NPL 1280 were most sig- duced copious amounts of EPS, 193.7 mg/L. EPS pro- nificantly antagonistic. Table 5 showed that all the duction was the least in P. pentosaceus strain NPL 1291 strains could co-aggregate with the pathogens tested, al- (88.91 mg/L). Moreover, all the L. plantarum strains dis- though the magnitude varied from strain to strain. L. played intermediate proteolytic activity (FAA 1 to 2 plantarum strains NPL 1258 and P. pentosaceus NPL mmol/L), whereas P. pentosaceus strains exhibited low 1264 exhibited the highest coaggregation with all patho- proteolytic activity (FAA < 1 mmol/L). The maximum gens tested (63–97%) following a 4-h incubation period. proteolytic activity value (1.89 ± 0.11 mmol/L) was All strains demonstrated deconjugation with sodium found for L. plantarum NPL 1258, whereas the mini- tauroglycocholate (TGC) and sodium taurocholate (TC) mum value (0.55 ± 0.02 mmol/L) was seen in P. pentosa- salts with varying substrate affinity. In general, most ceus strain NPL 1291 (Table 5). strains showed the least preference towards deconjuga- All the tested strains can reduce the nitrite concentra- tion of sodium taurodeoxycholate (TDC) compared with tion (Table 5). The highest sodium nitrite depletion rates other bile salts (Table 4). were exhibited by the L. plantarum strains NPL 1258 Cholesterol concentrations in the cell culture medium (75%), P. pentosaceus NPL 1264 (69%), followed by L. decreased after the initial solution was co-cultured with plantarum NPL 1284 (65%). However, the lowest deg- all the Lactobacillus strains (Table 5). Among the tested radation rates were observed for L. plantarum, NPL strains, NPL 1258 exhibited the highest cholesterol re- 1286 (18%). These results show that most of the tested moval rates (44%). The strains NPL 1279 and NPL 1291 strains are highly effective in depleting sodium nitrite. showed weaker rates of cholesterol removal (12% and Growth in 2% NaCl was interpreted as strong when it 19%, respectively). approximated average growth (without NaCl), which was All Lactobacillus strains exhibited antioxidative activity true for several strains (6 out of 10) (Table 3). Growth in (Table 5). The scavenging rates for hydroxyl radicals the presence of 4% NaCl was typically halved by 50%. ranged from 38 to 84%, and those for superoxide anion However, several strains of L. plantarum exhibited de- radicals ranged from 44 to 85.5%. NPL 1258 strain tectable growth at concentrations as high as 7% NaCl. In showed the highest capacity to scavenge hydroxyl radi- the present study, L. plantarum strain NPL 1259 was cals (84%), and NPL 1259 showed the highest capacity most robustly saline tolerant, withstanding 2%, 4%, and to scavenge superoxide anion radicals (85.5%). 7% NaCl by achieving 81%, 72%, and 65 % respectively L. plantarum strains NPL 1258, NPL 1284, and NPL of a typical growth profile. 1286 and P. pentosaceus strain NPL 1264 can utilize a multitude of sugar moieties such as monosaccharides, Multivariate analysis of the most suitable starter culture oligosaccharides, and some trisaccharides (raffinose), Principal component analysis was used to single out the along with some sugar alcohols (sorbitol, xylitol, and most promising starter culture for carrying out directed mannitol) and glycosides (esculin and salicin). Our Lacto fermentation of cucumber. The biplot graphs on strains are unable to metabolize disaccharides (maltose, PCA analysis are presented in Fig. 4. The first two fac- lactose, and melibiose) and deoxy sugars (fucose and tors represented 49.83% of the variability. From PCA rhamnose) (Table 6). Malonate utilization seems to be analysis, L. plantarum strains NPL 1258 and NPL 1280 absent in all L. plantarum and P. pentosaceus strains. and P. pentosaceus strain NPL 1264 were more associ- The select L. plantarum strains and P. pentosaceus ex- ated with starter culture and probiotic potential charac- hibited peptidase and esterase lipase (C8) activities. Al- teristics selected as a suitable candidate for lactic acid kaline phosphatase, esterase lipase (C4, C14), trypsin, fermentation of cucumber. These three strains were fur- and α-mannosidase and α-fucosidase were not active in ther tested for their compatibility with each other for the selected strains, nor activities of enzymes such as α- mixed culture through agar diffusion assay and cross- chymotrypsin, α-galactosidase, β-glucuronidase, N- streak assay. No inhibition halos of L. plantarum strain acetyl-β-glucosaminidase, N-acetyl-β-glucosaminidase NPL 1258 cell-free supernatants against the P. pentosa- activities deemed undesirable were recorded (Table 6). ceus NPL 1264 were observable, suggesting the absence Ahmed et al. Annals of Microbiology (2021) 71:33 Page 9 of 23 Table 2 Safety assessment of the LAB strains Strain Antibiotic susceptibility/ antibiotic resistance genes Hemolytic Gelatinase Biogenic amine code activity activity production AM/ GM/ KM/ SM/(aadA, EM/ CM/ TC/(tetM, CL/ His Orn Lys Tyr bla aac(6′)- aph(3′)- aadE, ant(6)) erm(A), (InuA, tetK, tetL) (catA, aph(2″) IIIa erm(B) InuB) cat) NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- - - - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ +- + - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ ++ - - + NPL S/- S/- S/- S/- S/- S/- S/- S/- α -+ - + - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - + - NPL S/- S/- S/- S/- S/- S/- S/- S/- α -- - + + NPL438 S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- S/- S/- S/- S/- S/- α -- + - - NPL S/- R/+ S/- R/+ S/- S/- S/- R/+ γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ + - + NPL S/- S/- S/- R/+ S/- S/- R/+ S/- γ -- - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- α +- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL S/- R/+ S/- S/- S/- S/- R/+ S/- β +- - - - Ahmed et al. Annals of Microbiology (2021) 71:33 Page 10 of 23 Table 2 Safety assessment of the LAB strains (Continued) Strain Antibiotic susceptibility/ antibiotic resistance genes Hemolytic Gelatinase Biogenic amine code activity activity production AM/ GM/ KM/ SM/(aadA, EM/ CM/ TC/(tetM, CL/ His Orn Lys Tyr bla aac(6′)- aph(3′)- aadE, ant(6)) erm(A), (InuA, tetK, tetL) (catA, aph(2″) IIIa erm(B) InuB) cat) NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - NPL R/+ S/- S/- R/+ S/- S/- S/- S/- γ -- - - - NPL S/- R/+ S/- S/- S/- S/- R/+ S/- β +- - - - NPL R/+ S/- S/- R/+ S/- S/- S/- S/- γ -- - - - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -+ - + - NPL S/- S/- S/- S/- S/- S/- S/- S/- γ -- - - - AM ampicillin, GM gentamycin, KM kanamycin, SM streptomycin, EM erythromycin, TC tetracycline, CM clindamycin, CL chloramphenicol, His histidine, Orn, ornithine, Lys lysine, Tyr tyrosine. Results are means of three independent experiments with three repetitions ± SD (n = 3). Table 3 Probiotic potential characteristics of the selected LAB strains Strain pH tolerance (%) Bile tolerance (%) Hydrophobicity Autoaggregation (%) NaCl Lysozyme Phenol code (%) tolerance resistance resistance (%) (0.4%) 1.5 3 0.15 0.30 Xylene t t t t 247 10 2 4 6 24 NPL 45.07 ± 73.24 ± 70.22 ± 38.89 ± 54 ± 0.02 20 ± 32 ± 46 ± 87 ± +++ - + + 1258 0.01 0.1 0.04 0.03 0.02 0.01 0.12 0.01 NPL 49.86 ± 62.43 ± 62.35 ± 27.41 ± 27 ± 0.01 11 ± 15 ± 18 ± 25 ± +++ - + + 1259 0.03 0.02 0.03 0.12 0.01 0.01 0.02 0.01 NPL 44.74 ± 67.01 ± 73.33 ± 38.18 ± 30 ± 0.04 13 ± 19 ± 28 ± 33 ± +++ - + + 1264 0.05 0.03 0.02 0.04 0.03 0.04 0.03 0.11 NPL 46.67 ± 73.33 ± 49.02 ± 13.73 ± 22 ± 0.0 28 ± 36 ± 50 ± 88 ± ++- - + + 1277 0.01 0.05 0.01 0.03 0.0 0.0 0.04 0.03 NPL 20.18 ± 46.49 ± 43.31 ± 19.75 ± 41 ± 0.11 34 ± 39 ± 43 ± 72 ± ++- - + + 1279 0.04 0.03 0.06 0.07 0.01 0.02 0.03 0.05 NPL 16.67 ± 25 ± 46.36 ± 23.84 ± 45 ± 0.06 10 ± 17 ± 26 ± 59 ± ++- - + + 1280 0.02 0.05 0.07 0.02 0.03 0.01 0.02 0.06 NPL 25 ± 31.25 ± 31.25 ± 13.75 ± 14 ± 0.05 27 ± 39 ± 53 ± 99 ± +++ - + + 1284 0.01 0.02 0.03 0.04 0.04 0.04 0.06 0.01 NPL 33 ± 87 ± 27.27 ± 19.39 ± 16 ± 0.02 12 ± 24 ± 37 ± 98 ± +++ - + + 1286 0.03 0.05 0.02 0.01 0.03 0.05 0.03 0.00 NPL 33.68 ± 51.58 ± 57.58 ± 34.09 ± 34 ±0.01 10 ± 22 ± 33 ± 67 ± +++ - + + 1291 0.02 0.06 0.01 0.03 0.02 0.01 0.0 0.02 NPL 30.83 ± 34.72 ± 59.64 ± 13.36 ± 31 ± 0.0 12 ± 19 ± 38 ± 65 ± ++- - + + 1306 0.03 0.03 0.02 0.02 0.01 0.02 0.02 0.01 ATCC 41.02 ± 66.31 ± 61.21 ± 25.42 ± 35 ± 0.11 17 ± 36 ± 41 ± 54 ± ++- - + + 8014 0.01 0.08 0.01 0.03 0.03 0.02 0.02 0.01 Results are means of three independent experiments with three repetitions ± SD (n = 3). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 11 of 23 Lacto-fermentation of cucumber using select starter strains Lactiplantibacilllus plantarum and Pediococcus pentosaceus driven cucumber fermentation prevents spoilage Plating count determined the microbial changes in dif- ferent groups during the fermentation. In all controlled fermented cucumber samples, LAB strains were the pre- vailing microorganisms throughout the process, and the maximum population of the inoculated strain was in treatment F (Fig. 5). The initial salt and inulin concen- trations of 4% (w/v) and 0.2% respectively in the treated sample favored rapid growth of mixed strains. LAB population was 8.3 log10 cfu/mL on the sixth day of fer- mentation which only reduced slightly by the 18th day. Whereas in other treatments, bacterial viability was sig- Fig. 2 Prebiotic utilization by LAB strains during growth. 1258: L. nificantly reduced to 7 log10 cfu/mL till the 18th day of plantarum, 1259: L. plantarum, 1264: P. pentosaceus, 1277: L. fermentation. Yeast and other pathogenic bacteria were plantarum, 1280: L. plantarum, 1284: L. plantarum, 1286: L. plantarum, observed in the control group after the sixth day. The 1291: P. pentosaceus, 1301: L. plantarum. Results are means of three number of LAB is also significantly reduced after the 6th independent experiments with three repetitions ± SD, n =3 day of fermentation. Defined lacto-fermentation of cucumber driven by the high of antimicrobial substances that could prevent using lactic acid content these strains in mixed cultures. However, the limited On the first day of fermentation, the pH value of fer- growth of L. plantarum strains NPL 1280 was observed mented samples ranged from 3.9 to 4.4. Following 18 against P. pentosaceus NPL 1264. The cross-streak assay days of fermentation, the pH of the brine solutions de- showed similar results, as no evident competition was creased from 3.5 to 3 (Fig. 5). The pH drop in all treat- noticeable at sites of co-growth in a solid medium in ments was highly significant (P < 0.05) between the combination with NPL 1258 and NPL 1264, allowing control sample and others, but there were no significant their potential use as a mixed starter (results not differences among treatments during fermentation. The shown). pH drop was sharpest in treatments E and F and then plateauing to levels like others. The decrease of pH values in A and B samples were slighter than others. Lactic acid (g/100 mL) production continually increased in all inoculated samples relative to the control. Acetic acid (g/100 mL) production was negligible in all treat- ment samples except the control sample, where it spiked at the end. Sensory acceptability of lacto-fermented cucumber Sensory evaluation of fermented cucumber samples was performed at the culmination of the process using a panel of 10 non-trained persons. For flavor, the panelists gave an overall higher rank to samples made using both candidate LAB starter strains plus inulin than all other treatments (Fig. 6). The crunchiness was best appreci- ated in products made using dual than mono starters. LAB strains invariably contribute to the aroma, texture, and flavor of fermented products. The sharp increase in Fig. 3 In vitro digestion assay of the selected Lactobacilli without (a) acidity minimized the influence of spoilage bacteria and and with (b) food matrix. 1258: L. plantarum, 1259: L. plantarum, 1264: P. pentosaceus, 1277: L. plantarum, 1280: L. plantarum, 1284: L. consistently improved the microbiological and sensory plantarum, 1286: L. plantarum, 1291: P. pentosaceus, 1301: L. quality of the fermented product than would be possible plantarum. Results are means of three independent experiments in fermentations without defined starters (Tamang and with three repetitions ± SD, n =3 Tamang 2010). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 12 of 23 Table 4 Functional properties of selected LAB strains * ɷ ɣ Strain Diameter of inhibition zone (mm) Deconjugation of bile salts Decarboxylation of phenolics code S. p S. a E. c B. c P. a C. f TGC TC TDC Tann Gall NPL 1258 +++ +++ +++ ++ ++ ++ + + + + + NPL 1259 ++ ++ ++ ++ +++ ++ + + - + + NPL 1264 +++ ++ +++ ++ +++ ++ + + - + + NPL 1277 ++ ++ +++ ++ ++ ++ - + - + + NPL 1279 +++ ++ ++ ++ ++ ++ + + - + + NPL 1280 + + +++ + + + + + - + + NPL 1284 +++ +++ +++ ++ ++ ++ + + + + + NPL 1286 ++ ++ ++ ++ ++ ++ + + - + + NPL 1291 + + ++ ++ + ++ + + - + + NPL 1306 ++ +++ ++ ++ +++ ++ + + - + + ATCC 8014 ++ ++ ++ ++ ++ ++ + + - + + S. p, Streptococcus pyogenes ATCC 19615; S. a, Staphylococcus aureus ATCC 25923, E. c, Escherichia coli ATCC 25922; B. c, Bacillus cereus ATCC 11778; P. a. Pseudomonas aeruginosa ATCC 15442; C. f. Citrobacter freundii ATCC 8090 * (+) weak (< 10 mm), (++) intermediate (10–20 mm), (+++) strong (< 20 mm), (−) no zone TGC, tauroglycocholate; TC, sodium taurocholate; TDC, sodium taurodeoxycholate; DC, sodium deoxycholate (+) present, (-) absent. Results are means of three independent experiments with three repetitions ± SD (n =3) Table 5 Functional properties of selected LAB strains Strain Antioxidant activity Cholesterol Nitrite Proteolytic EPS Coaggregation (%) code assimilation degradation activity (mg/L) Hydroxyl Superoxide S. a S. p E. c B. c P. a (%) (%) (mmol/L) radical (%) anions (%) NPL 84 ± 0.22 64.65 ± 0.17 44 ± 0.12 75 ±0.01 1.89 ± 0.11 85.5 ± 77 ± 85 ± 66 ± 52 ± 52 ± 1258 0.01 0.11 0.05 0.02 0.01 0.01 NPL 48 ± 0.12 85.5 ± 0.22 38 ± 0.02 59 ± 0.12 1.73 ± 0.0 74.9 ± 31 ± 43 ± 87 ± 41 ± 41 ± 1259 0.12 0.03 0.08 0.04 0.03 0.03 NPL 73 ± 0.14 64.9 ± 0.32 33 ± 0.11 69 ± 0.05 0.76 ± 0.0 92.1 ± 97 ± 66 ± 53 ± 63 ± 63 ± 1264 0.15 0.06 0.13 0.04 0.04 0.04 NPL 52 ± 0.31 46.15 ± 0.45 22 ± 0.21 36 ± 0.03 1.61 ± 0.16 86.8 ± 34 ± 82 ± 62 ± 47 ± 47 ± 1277 0.17 0.13 0.16 0.03 0.5 0.5 NPL 60 ± 0.11 76.85 ± 0.27 12 ± 0.12 55 ± 0.17 1.38 ± 0.09 88.6 ± 74 ± 44 ± 36 ± 54 ± 54 ± 1279 0.21 0.16 0.04 0.11 0.11 0.11 NPL 56 ± 0.16 68.65 ± 0.22 29 ± 0.05 65 ± 0.02 1.41 ± 0.05 76.1 ± 87 ± 76 ± 55 ± 48 ± 48 ± 1280 0.33 0.25 0.02 0.03 0.21 0.21 NPL 74 ± 0.15 76.31 ± 0.13 31 ± 0.02 56 ± 0.14 1.60 ± 0.02 85.2 ± 29 ± 22 ± 48 ± 63 ± 63 ± 1284 0.12 0.15 0.05 0.02 0.04 0.04 NPL 66 ± 0.06 84.18 ± 0.17 23 ± 0.16 18 ± 0.11 1.67 ± 0.11 71.9 ± 43 ± 52 ± 58 ± 33 ± 33 ± 1286 0.11 0.01 0.11 0.01 0.04 0.04 NPL 53 ± 0.11 64.98 ± 0.19 19 ± 0.41 58 ± 0.04 0.55 ± 0.02 88.91 ± 63 ± 73 ± 61 ± 16 ± 16 ± 1291 0.02 0.00 0.05 0.01 0.2 0.2 NPL 38 ± 0.09 44.34 ± 0.32 20 ± 0.01 34 ± 0.01 1.52 ± 0.03 91 ± 75.3 ± 58 ± 49 ± 82 ± 64 ± 1306 0.04 0.02 0.21 0.12 0.01 0.01 ATCC 44 ± 0.03 62.34 ± 0.02 25 ± 0.04 56 ± 0.02 0.92 ± 0.01 83 ± 66.78 ± 67 ± 56 ± 71 ± 39 ± 8014 0.01 0.2 0.11 0.11 0.01 0.02 S. p, Streptococcus pyogenes ATCC 19615; S. a, Staphylococcus aureus ATCC 25923, E. c, Escherichia coli ATCC 25922, B. c Bacillus cereus ATCC 11778. P. a Pseudomonas aeruginosa ATCC 15442. Results are means of three independent experiments with three repetitions ± SD (n = 3). Ahmed et al. Annals of Microbiology (2021) 71:33 Page 13 of 23 Table 6 Enzyme characterization and carbohydrate utilization of selected LAB NPL NPL NPL NPL NPL NPL NPL NPL NPL NPL ATCC 1258 1259 1264 1277 1279 1280 1284 1286 1291 1306 8014 Sr. Carbohydrate no. fermentation 1 Lactose + + + + + + + + + + + 2 Xylose + + + + + + + + + + + 3 Maltose + + - + + + + + - + + 4 Fructose + - + + - + + + + + + 5 Dextrose + - + + - + + - + + + 6 Galactose + - + + - + + - + + + 7 Raffinose + - + + - + + + + - - 8 Trehalose + + + + + + + + + + + 9 Melibiose - - - - - - - - - - - 10 Sucrose + - + + - + + + + + + 11 L-Arabinose + + + + + + + + + + + 12 Mannose + - + + - + + + + + + 13 Inulin + + + + + + + + + + + 14 Sodium gluconate - - - - - - - - - - - 15 Glycerol - - - - - + + - - - - 16 Salicin + + + + + + + + + - + 17 Dulcitol - - - - - - + - - - - 18 Inositol - - - - - - - - - - - 19 Sorbitol + + + + + + + + + + + 20 Mannitol + + + + + + + + + + + 21 Adonitol - - - - - - + - - - + 22 Arabitol - - - - - - + - - + + 23 Erythritol - - - - - - - - - - - 24 α-Methyl-D glucoside - - - - - - + - - - - 25 Rhamnose - - - - - - + - - - - 26 Cellobiose + + + + + + + + + + + 27 Melezitose - + - - - - + - - - - 28 α-methyl-D mannoside + + - - - - + - - - - 29 Xylitol + + - - - - + - - - - 30 ONPG + + - - - - + - - - - 31 Esculin hydrolysis + + + + + + + + + + + 32 D-Arabinose + + + + + + + + + + + 33 Citrate utilization - + - - - + + + - - + 34 Malonate utilization - + - - - - - - - - - 35 Sorbose - - - - - - - - - - - Sr. Enzyme activity no. 1 Alkaline phosphatase 0 0 0 0 0 0 0 0 0 0 0 2 Esterase (C 4) 0 0 0 0 0 0 0 0 0 0 0 3 Esterase lipase (C 8) 5 5 10 5 10 10 10 5 10 5 5 4 Lipase (C 14) 0 0 0 0 0 0 0 0 0 0 0 5 Leucine arylamidase > 40 20 20 20 > 40 > 40 > 40 20 10 10 > 40 Ahmed et al. Annals of Microbiology (2021) 71:33 Page 14 of 23 Table 6 Enzyme characterization and carbohydrate utilization of selected LAB (Continued) NPL NPL NPL NPL NPL NPL NPL NPL NPL NPL ATCC 1258 1259 1264 1277 1279 1280 1284 1286 1291 1306 8014 6 Valine arylamidase 20 20 20 20 20 20 20 20 5 5 10 7 Cystine arylamidase > 40 > 40 20 20 20 20 20 20 20 20 20 8 Trypsin 0 0 0 0 0 0 0 0 0 0 0 9 α-chymotrypsin 0 0 0 0 0 0 0 0 0 0 0 10 Acid phosphatase 5 5 5 5 5 5 5 5 5 5 10 11 Naphthol-AS-BI- 5 555 555 555 20 phosphohydrolase 12 α-galactosidase 0 0 0 0 0 0 0 0 0 0 0 13 ß-galactosidase 20 20 > 40 20 20 > 40 20 20 20 20 20 14 ß-glucuronidase 0 0 0 0 0 0 0 0 0 0 0 15 α-glucosidase 0 0 0 0 0 0 0 0 0 0 0 16 ß-glucosidase 20 10 20 10 5 5 5 10 5 10 20 17 N-acetyl-ß-glucosaminidase 0 0 0 0 0 0 0 0 0 0 0 18 α-mannosidase 0 0 0 0 0 0 0 0 0 0 0 19 α-fucosidase 0 0 0 0 0 0 0 0 0 0 0 Enzyme activities were assessed through API-ZYM galleries that were expressed in terms of color intensity, which ranged from 0 (no activity), 5–10 (low), 30 (moderate), and ≥ 40 nmol (strong) of substrate hydrolyzed following 4 h incubation at 37 °C. Color intensity was judged with reference to an API ZYM color chart provided by the manufacturer. ‘+’ shows a positive reaction; ‘−’ shows a negative reaction Fig. 4 PCA biplot projecting the probiotic potential starter culture variables and strains Ahmed et al. Annals of Microbiology (2021) 71:33 Page 15 of 23 Fig. 5 Microbiological and physicochemical analysis of lacto-fermented cucumber during fermentation period. a Changes in viable cell number of LAB in different samples, b pH, c LA (Lactic acid), and d AA (Acetic acid) during fermentation. Control: without bacterial inocula with 4% (w/v) NaCl, pH 4; A: L. plantarum NPL 1258 with 4% (w/v) NaCl, pH 4; B: L. plantarum NPL 1258 with 4% (w/v) NaCl, 0.2% inulin, pH 4; C: P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; D: P. pentosaceus NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4; E: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; F: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4. Results are means of three independent experiments with three repetitions ± SD, n =3 Discussion Fermented cucumber is a popular culinary choice in many Asian countries (Behera et al. 2020). Fermented vegetables containing LAB are being increasingly researched because of their benefits to vegetarians and individuals with dairy intolerances (Di Cagno et al. 2013). Many vegetables with good nutritional content but otherwise poor organoleptics can be rendered flavor- ful and desirable for consumption when fermented by LAB species (Alan et al. 2018). Autochthonous LAB abounds spontaneously fermentation vegetables, an es- sentially stochastic event buffeted by numerous environ- mental parameters (Xu et al. 2018). A slew of potential autochthonous starter cultures must be examined and the right fit identified to standardize and control the Fig. 6 Sensory analysis of lacto-fermented cucumber after fermentation period. Control: without bacterial inocula with 4% (w/ process, especially in large-scale cucumber fermentations v) NaCl, pH 4; A: L. plantarum NPL 1258 with 4% (w/v) NaCl, pH 4; B: (Jampaphaeng et al. 2018). L. plantarum NPL 1258 with 4% (w/v) NaCl, 0.2% inulin, pH 4; C: P. 2 3 Generally, LAB constitute a small part (10 –10 cfu/g) pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; D: P. pentosaceus of the full spectrum of autochthonous microbiota of raw NPL 1264 with 4% (w/v) NaCl, 0.2% inulin, pH 4; E: L. plantarum NPL vegetables (Kothari et al. 2020). Lactobacillus spp., Leu- 1258 and P. pentosaceus NPL 1264 with 4% (w/v) NaCl, pH 4; F: L. plantarum NPL 1258 and P. pentosaceus NPL 1264 with 4% (w/v) conostoc spp., and Pediococcus spp. are the leading LAB NaCl, 0.2% inulin, pH 4. Results are means of three independent predominating on the cucumber surface and are respon- experiments with three repetitions ± SD, n =3 sible for fermenting it (Behera et al. 2020). Finding Ahmed et al. Annals of Microbiology (2021) 71:33 Page 16 of 23 plentiful L. plantarum is expected since it is the pre- (Benítez-Cabello et al. 2019) and global regulatory guide- dominant Lactobacillus associated with SF cucumber be- lines emphatic stance on excluding probiotic candidates cause of its penchant for thriving in high acidity and with even a smidgen of hemolytic behavior (Food et al. salinity (Behera et al. 2018). 2006). Enzyme profiling is also helpful for selecting A multitude of LAB spp. such as L. brevis, S. thermo- strains to be used in food, as their enzymatic activities philus, E. faecium, P. pentosaceus, and Leuconostoc may affect the quality of the product (Colombo et al. mesenteroides in association with the fermented vege- 2018). table matter as seen here is in line with the notion where In recent years, it has been posited that food bacteria a broad vegetal microbial diversity could be attributed to may act as reservoirs of antibiotic resistance genes, a slew of agro-technological factors such as cultivars, which might be transferred to gut commensals or patho- geography, seasons, and aspects of the fermentation gens (Hummel et al. 2007). However, Lactobacillus, es- process like the quality of the base material used and the pecially those of fermented food origin, are unlikely to equipment surfaces (Khalil et al. 2018). Leuconostoc contribute to the AMR (Antimicrobial Resistance) prob- mesenteroides is an infrequent primary fermenter of cu- lem in humans (Ma et al. 2017). Nevertheless, the Euro- cumber whose numbers are eventually superseded by L. pean Food Safety Authority (EFSA) considers its plantarum, which finishes the process (Fusco et al. assessment a primary criterion for according “qualified 2017). A greater abundance of Enterobacteriaceae was presumption of safety” (QPS) status (Zielińska et al. observed on conventionally farmed vegetables than or- 2015). Intrinsic resistance to antibiotics such as amino- ganically farmed via culturing technique (Leff and Fierer glycosides and vancomycin in Pediococcus (Shukla and 2013). This effect could be attributed to several factors: Goyal 2014) and Lactobacillus species such as L. plan- growing location, fertilizer use, pesticide use, other agri- tarum are intrinsic, non-transferable, and not sufficiently cultural practices, and shipping and handling procedures concerning to prevent their use for food fermentation (Beuchat et al. 2006). Enterococcal species as E. hirae, E. (Abriouel et al. 2015; EFSA 2018). Therefore, all strains faecium, and E. cloacae, although naturally present on carrying antibiotic-resistant genes were adjudged as un- cucumber, are pinned down during the LAB-driven pri- safe and precluded from further study. A high incidence mary fermentation phase, only rebounding when the pH of antibiotic resistance in Lactobacillus species has been starts to rise. Since E. cloacae tends to spoil fermenting attributed to insufficient checks and balances for the use cucumbers, its presence is considered undesirable and disposal of antibiotics in developing societies (Ma (Franco and Pérez-Díaz 2013). et al. 2017). Before assaying for probiotic functionality in candidate The probiotic effectiveness of these innocuous candi- strains, their safety status was determined in vitro as rec- date starters was gauged by testing their tolerance to hu- ommended by FAO (Food et al. 2006) since probiotic man gut physiological parameters such as pH, and bile starter cultures are consumed live in large dosages tolerance must be examined in vitro (Anandharaj et al. through the fermented product; therefore, their harm- 2015). Such an approach is faster, cheaper, reproducible, lessness to public health must be ensured (Chokesajja- and offers more choice of controlled conditions disen- watee et al. 2020). LAB spp. generally have a good safety cumbered with many ethical restrictions associated with record for human consumption; however, there are some in vivo experimentation (Calvo-Lerma et al. 2019). exceptions involving L. plantarum (Cannon et al. 2005) Robust acid tolerance in a potential probiotic starter and the enterococcal species E. faecium and E. faecalis candidate, besides empowering it to survive the human (Sanchez Valenzuela et al. 2013; Strateva et al. 2016). host’s GIT milieu, also prolongs its survivability in highly LAB that are amino biogenic during spontaneous lactic acidic fermented foods (Michalak et al. 2020). Tolerating acid fermentation can jeopardize the quality and safety extreme acidity as seen in L. plantarum strains NPL of fermented foods (Alan et al. 2018), thus are ill-suited 1258, NPL 1259, and NPL 1280 is likely due to their for use as starters and adjuncts (Behera et al. 2020; Beli- adaptiveness to the pickle habitat (Adebayo-tayo and cová et al. 2013). Both candidate starters and probiotics Onilude 2008) from where they have been isolated in must incontrovertibly demonstrate an absence of harm- this study. Withstanding bile exposure is vital for an ful enzymatic activities, such as β-glucosidase and β- ingested probiotic to survive in the small intestine (Ana- glucuronidase, known to cause detrimental effects in the ndharaj et al. 2015). Tolerating 0.15–0.3% concentration colon (Zielińska et al. 2015). Testing for virulence attri- of bile salts is a sufficient threshold for any probiotic butes such as hemolysins, typically associated with path- taken orally (Alp and Aslim 2010). The extent to which ogens, was also deemed essential because of a past Lactobacilli can withstand bile is crucial since its levels precedent of its occurrence, albeit a sporadic one in in the gut are not static, fluctuating from 1.5 to 2% (w/v) some Lactobacillus species (Domingos-Lopes et al. in the first hour of digestion and eventually plateauing 2017), including L. plantarum strains of vegetable origin out at 0.3% (w/v) (Bao et al. 2010). LAB strains of Ahmed et al. Annals of Microbiology (2021) 71:33 Page 17 of 23 vegetable origins are generally less bile resistant (Chiu autoaggregative strength (Lee et al. 2014). However, the et al. 2008), contrasting with our findings where some L. extent to which cell hydrophobicity correlates with self plantarum strains were significantly bile resistant. The and coaggregation can vary in LAB spp. (Li et al. 2015). adaptation mechanism to bile salts is multifactorial. It is Coaggregation is advantageous because it allows a pro- attributed to changes in the bacterium’s ability to fer- biotic strain to produce antimicrobial substances in ment carbohydrates, exopolysaccharides production, the proximity to several pathogen cells, effectively eliminat- balance of proteins and fatty acids in its cell membrane, ing them from the GIT (Tuo et al. 2013). and the ability to firmly adhere to human mucus (Ali Among the technological properties much sought in et al. 2020). LAB are EPS production, salt tolerance, and antimicro- Any prospective probiotics added to a food matrix bial metabolites. The ability of LAB to produce EPS is a should tolerate food processing and storage conditions common trait of LAB starters as it helps to improve the and the entire swathe of gastrointestinal transit following colonization of probiotic bacteria in the gastrointestinal ingestion right up to their intestinal site of action (Cam- tract and protect against the baleful effects of harmful pos et al. 2019). A high titer of probiotic bacteria in food resident bacteria (Kumar et al. 2017). Our finding of products at the point of consumption does not guaran- copiously EPS-producing L. plantarum strains from fer- tee the same numbers in the gut because stomach acid- mented cucumber has some precedent (Jiang et al. ity and intestinal bile can dramatically reduce their 2016). viability (da Cruz Rodrigues et al. 2019). Estimating the The antimicrobial activity of LAB may improve the ravages of a GIT transit can be best done by simulating quality of fermented foods by eliminating spoilage and digestive processes in vitro (Campos et al. 2019), starting pathogenic bacteria (Michalak et al. 2020). Our results from the mouth to the ileum, and factoring in the effects align with previous findings where L. plantarum strains of the food matrix, enzymes, and peristalsis (Neffe-Sko- inhibited gram-positive bacteria such as S. aureus, S. cińska et al. 2018). Aside from its simplicity, low cost, pyogenes, and B. cereus, and Gram-negative C. freundii, and high throughput format, the clincher in this method E. coli, and P. aeruginosa (Gheziel et al. 2019; Jiang et al. is sequential exposure to acid and bile, a more authentic 2016). Indeed, antagonistic activity against E. coli is a depiction of actual physiological events. Our findings of relevant screening criterion because of the frequent L. plantarum and Pediococcus strains capable of with- presence of coliforms in cucumber pickle brine (Lu et al. standing GIT transit without any significant deleterious- 2013). Other food quality indicator microorganisms are ness support previous reports (Barbosa et al. 2015; also essential to test due to their high load on cucumber Gheziel et al. 2019). L. plantarum derived from SF cu- surfaces. Pediococcus species such as P. pentosaceus are cumber are likely to be intrinsically strong acid-tolerant inhibitory of pernicious and ubiquitous human patho- because the pickled vegetables mimic harsh gastrointes- gens such as L. monocytogenes, which has been challen- tinal conditions with pH values reaching three or lower ging to control with standard industrial approaches (Cao et al. 2019). (Huang et al. 2009). One of the caveats of a good probiotic is that the Since cucumber fermentation occurs in brine with 5– strain must also endure the action of toxic metabolites 7% NaCl (Di Cagno et al. 2008), candidate LAB starter (primarily phenols) produced during the digestion strains ought to be halotolerant, as is the case here process (Jawan et al. 2019), which is also observed here. (Rodriguez-Palacios et al. 2017). Some strains of L. plan- The selected L. plantarum and P. pentosaceus strains tarum were exceptionally tolerant of salt, which may be harbored a significant ability to metabolize food pheno- due to the accumulation of osmo and cryoprotective sol- lics via active tannase and gallate decarboxylases, de- utes such as betaine and carnitine (Yao et al. 2020). grading tannin and gallic acids. Both are anti-nutritional Halotolerance is not a staple characteristic of the species factors (ANF) that inactivate digestive enzymes, as much variability is observed (Ziadi et al. 2019). insolubilize proteins, and affect the utilization of vita- Carbohydrate utilization by lactic acid bacteria (LAB) mins and minerals (Sáez et al. 2018). Microbial metabo- defines the extent of cucumber fermentations, their lization of nitrite during cucumber fermentation is also quality, and long-term stability. Aside from glucose and advantageous since dietary nitrite is linked to gastro- fructose, alternate energy sources such as trehalose, cel- intestinal cancers (Behera et al. 2020), and its control is lobiose, and xylose, available in cucumber fermentations. important from a food safety standpoint (Ren et al. These compounds remained present in cucumber fer- 2014). mentations even after glucose and fructose were con- LAB cells that strongly aggregate and whose surfaces sumed. The removal of these alternate energy sources by are hydrophobic can adhere well to intestinal epithelia starter cultures during the most active period of the bio- and occlude gut luminal pathogens (Abbasiliasi et al. conversion prevents the proliferation of spoilage- 2017). BATH values are usually a proxy for associated microbes such as L. buchneri (Ucar et al. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 18 of 23 2020a; Ucar et al. 2020b). The strains of L. plantarum digestibility (Rizzello et al. 2016). A weakly proteolytic and P. pentosaceus used in this study can use trehalose behavior of LAB species found in this study could be and cellobiose and hamper the growth and metabolic ac- tied to their vegetal origins that are poor in protein con- tivity of spoilage-associated microbes. Our L. plantarum tent (Sáez et al. 2018). and P. pentosaceus strains can utilize plant-based carbo- Bile salt hydrolase (BSH) activity is essential for bac- hydrates such as mannitol and inulin (Gustaw et al. teria to thrive in the intestine and for the human host 2018; Lee et al. 2014), which helps to promote the who can benefit from its cholesterol-lowering effect growth of Lactobacillus. The raffinose-degrading ability (Jones et al. 2013). BSH hydrolyzes conjugated glyco- of our strains is an appealing feature because of their as- deoxycholic and taurodeoxycholic acid into glyco- and sociation with flatulence and GI disturbances in humans tauro-bile acids, respectively (Anandharaj et al. 2015), (Arunraj et al. 2020). Malonate utilization seems absent thereby protecting gut microbiota and probiotic bacteria in all L. plantarum and P. pentosaceus strains associated from their toxicity (Shukla and Goyal 2014). Typically, with bloating during cucumber fermentation (Bintsis LAB with vegetal origins where bile salts are absent do 2018). Our strains’ diverse metabolic profile helps them not possess bile salt hydrolase activity (Zielińska et al. endure well in various non-dairy food matrices and the 2015) which is at odds with our finding. Nevertheless, human intestine (Gupta and Bajaj 2018). both L. plantarum and P. pentosaceus have been shown An extracellular enzyme produced by the candidate to have BSHs active against tauroconjugates of bile salts probiotics in the food matrix may improve the organo- (Lee et al. 2014). This study supports previous conten- leptic properties of food and enhance human digestion tions where LAB with active bile salt hydrolase has been (Gupta and Bajaj 2018). Enzymatic profiles of LAB that shown to lower cholesterol levels, removing secondary lack proteinases but have strong peptidase and esterase bile salts and cholesterol from the human body (Peres lipase activities are associated with imparting typical and et al. 2014). The extent to which they are successful is desirable flavors to fermented vegetable products (Abba- highly strain dependent (Zhang et al. 2014). siliasi et al. 2017; Goswami et al. 2017); therefore, the Inulin, fructooligosaccharides, and maltodextrins are presence of these traits in our strains confirms their es- well-established prebiotics that remain undigested in the sential role in flavor development of fermented cucum- upper gastrointestinal tract and are only fermented by ber. Acid phosphatase and phosphohydrolase allow the Lactobacillus in the colon (Choudhary et al. 2019). In- probiotic strains to metabolize diverse substrates of the corporating them into a probiotic carrying food matrix GIT (Shokryazdan et al. 2017). The absence of harmful should have a proliferative effect on probiotic LAB, enzymes, α-glucosidases or β-glucuronidase, implicated which is the case here. LAB species have cell-associated in carcinogenesis (Yeo et al. 2016), and chymotrypsin glycosidases and fructofuranosidases for hydrolysis of and N-acetyl-B-glucosaminidase that are linked with these substrates and using the resulting monomers as an GIT dysfunction (Delgado et al. 2007) renders strains energy source (Perrin et al. 2001). Choosing the right safer for human consumption. prebiotic is deemed essential and must be empirically Strains with copious EPS production with significant determined for any novel synbiotic to succeed. We chose halotolerance and anti-pathogen capabilities were also inulin instead of FOS as the prebiotic part of the synbio- assessed for withstanding oxidative stress. The antioxida- tic for lacto-fermentation of cucumber because the tive mechanisms protect colonizing LAB from attack by former was more readily fermentable and improved free radicals while benefiting the human host by mitigat- colonization and persistence of L. plantarum (Brajdes ing cardiovascular diseases, diabetes, and ulcers of the and Vizireanu 2013). The FOS, on the other hand, is GI tract (Ren et al. 2014). It was found that some strains more suitable as prebiotic to bifidobacterial instead of of L. plantarum registered more antioxidant capacity Lactobacillus spp. in synbiotic combinations (Schrezen- than the reference strain. Vegetable matrices are typic- meir and de Vrese 2001). ally characterized as high antioxidant environments When considering starters tailored for cucumber fer- (Verni et al. 2017). An aspect that has not gone un- mentation, the selection of strains from its natural noticed since fermented vegetables’ consumption was microbiota allows for by-passing the adaptation chal- found to correlate with a lower COVID-19 pandemic lenges that allochthonous cultures could face, thus facili- mortality rate (Fonseca et al. 2020). tating an improved nutritional, functional, and The proteolytic activity of LAB enhances the organo- technological profile of fermented cucumber (Verni leptic profile of fermented foods through the release of et al. 2017). L. plantarum and Pediococcus spp. are well- free amino acids and their derivatives (Karasu et al. recognized starter cultures giving various fermented 2010; Verni et al. 2017). In addition, they are also in- vegetable products (Behera et al. 2018). Although strains volved in the degradation of allergenic and anti- could be selected based on their technological features, nutritional proteins and the increased protein multiple attributes are more helpful in making foods that Ahmed et al. Annals of Microbiology (2021) 71:33 Page 19 of 23 have better sensory properties. A principal component acid through its exclusive homofermentative pathway analysis (PCA) can facilitate screening out the most suit- (Güney and Güngörmüşler 2020). The production of able strain for fermenting vegetables (Sáez et al. 2018). acetic acid was less than the lactic acid in all inoculated Mixed species inoculation of brined cucumbers with P. cucumber samples. The decreased concentrations of lac- pentosaceus and L. plantarum has the potential advan- tic acid accompanied by increased acetic acid concentra- tage of an early, rapid initial growth and moderate acid tions in the control fermented cucumber sample could production by the former species and a higher final acid- be due to the degradation of lactic acid into acetic acid ity resulting from the lower pH tolerance of the latter by spoilage-associated microorganisms, especially L. (Tamang and Tamang 2010). buchneri (Johanningsmeier and McFeeters 2013). The typical industry standard of a minimum of 10 Sensory analysis is an indispensable tool to determine cfu/g of probiotics in a product stems from clinical in- consumer acceptability (Cuffia et al. 2018). Irrespective 8 9 vestigations where a minimum of 10 –10 cfu/g of pro- of a product’s health claims, the customer will likely re- biotic was deemed necessary for health benefits (da Cruz ject it if the sensory profile is poor (Karimi et al. 2012). et al. 2009). Maintenance of appropriate numbers of We used an affective sensory method using a 5-point he- probiotics during the storage of fermented vegetables is donic scale popular in the industry and academic re- quite challenging due to the low pH of brine, nutrient search (Greifova 2007). The concept is serially monadic depletion, and the accumulation of lactic acid (Valero- that does not allow for retasting or contextual reference Cases and Frutos 2017). The supplementation of a nat- to capture consumer purchase behavior more accurately ural plant origin prebiotic such as inulin can stimulate (Wichchukit and O'Mahony 2015). Sensory analysis is a beneficial bacteria’s growth and metabolic activity in try- human-centric propriocentric view of a food product. ing conditions (Nilchian et al. 2016). It can also protect Taste and pleasure are among the most meaningful pre- the cells from refrigeration-associated cell damage, dictors of food choice (Brunsø et al. 2002). Lacto- mainly through physical immobilization of the cells in fermented cucumbers with mixed starter cultures were inulin macroaggregates (Bedani et al. 2013). Before fer- preferable to monoculture fermented cucumber con- mentation, the addition of prebiotics improves the shelf cerning taste and pleasure. This difference might be be- life viability of probiotic bacteria mixed in with a food cause mixed strain cultures are relatively less affected by product (Szydłowska and Kołożyn-Krajewska 2019). The vicissitudes of handling, storage, and applications and pro-proliferative and protective effect of inulin in L. contribute more to desirable flavors while cutting down plantarum seems strain-dependent judging from reports on unpleasant ones (Holzapfel 2002). Bitterness and over in its favor (Valero-Cases and Frutos 2015) and disfavor sourness are negatively hedonistic (Greifova 2007), and (Nazzaro et al. 2012). mar acceptance of control spontaneously fermented cu- Starters bring about a rapid decrease of pH, which cumbers (Drewnowski and Gomez-Carneros 2000; Ver- helps to reduce the risk of spoilage at the beginning of heul et al. 2013). fermentation (Nilchian et al. 2016). The ability to acidify rapidly is desirable for any would-be LAB starter of Conclusion vegetable fermentation (Wakil et al. 2014). The ability to The present study highlights the capacity of two LAB acidify a medium is typical of many LAB species because strains, autochthonous to SF cucumber, as potential pro- of organic acids, mainly lactic acid (Greifova 2007). L. biotic cum starter culture candidates. The results indi- plantarum directed acidification of the samples has been cate that inoculated selected starters manifested in vitro applied for food preservation (Muthusamy et al. 2020). several desirable, beneficial probiotic attributes such as Optimizing brine concentration empirically as done here antioxidant, BSH activity, cholesterol assimilation, and is critical for a desirable and appropriately fermented cu- antibiotic susceptibility. In addition, the inoculated cumber (Nilchian et al. 2016). Higher concentrations of starters remained significantly viable during fermenta- brine promote spoilage because of lowered pH (Bautista- tion and contributed to the aroma and flavor of the fer- Gallego et al. 2010). mented cucumber. They prevented putrescence caused Lactic acid and acetic acid are the primary metabolic by spoilage bacteria and enhanced the sensorial aspects end products of carbohydrate fermentation during cu- of the fermented product. A high inoculum of L. plan- cumber fermentation by LAB, which lowers the food tarum and P. pentosaceus strains (NPL 1258 and NPL pH, imbuing it with desirable organoleptic properties 1259) was found to control the fermented cucumber’s and eliminating pathogens, ensuring safety and stability quality effectively. Strain probioticity, no doubt a good of the final product (Adesulu-Dahunsi et al. 2018). A sig- and valuable attribute, nonetheless does not obviate the nificant increase in lactic acid production during anaer- need to examine the fermentation kinetics and physio- obic fermentation of cucumber is associated with the logical benefits further before they could be recom- capability of L. plantarum for making substantial lactic mended for large-scale commercial application. Ahmed et al. Annals of Microbiology (2021) 71:33 Page 20 of 23 Acknowledgments Anandharaj M, Sivasankari B, Santhanakaruppu R, Manimaran M, Rani RP, Not applicable Sivakumar S (2015) Determining the probiotic potential of cholesterol- reducing Lactobacillus and Weissella strains isolated from gherkins (fermented cucumber) and south Indian fermented koozh. Res Microbiol 166(5):428–439. Data availability https://doi.org/10.1016/j.resmic.2015.03.002 All data generated or analyzed during this study are included in this Arunraj R, Skori L, Kumar A, Hickerson NM, Shoma N, Samuel MA (2020) Spatial published article. regulation of alpha-galactosidase activity and its influence on raffinose family oligosaccharides during seed maturation and germination in Cicer arietinum. Authors’ contributions Plant Signal Behav 15(8):1709707. https://doi.org/10.1080/15592324.2019.1 The contributions of SA and AZ are equal. SA contributed to the investigation, data analysis, writing the original draft. FA contributed to the Bao Y, Zhang Y, Zhang Y, Liu Y, Wang S, Dong X, Wang Y, Zhang H (2010) investigation. MT did the validation, resources. AZ did the conceptualization, Screening of potential probiotic properties of Lactobacillus fermentum experimental design, writing, reviewing, editing, visualization, supervision, isolated from traditional dairy products. 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Annals of Microbiology – Springer Journals

Published: Dec 1, 2021

Keywords: Fermented cucumbers; Lactic acid bacteria; Functional properties; Inulin; Starter co-cultures

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Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

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Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus

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