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1. ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–9 ! 2013 Informa UK Ltd. DOI:…
  • 1. ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–9 ! 2013 Informa UK Ltd. DOI: 10.3109/09637486.2013.832175 RESEARCH ARTICLE Probiotic characterization of potential hydrolases producing Lactococcus lactis subsp. lactis isolated from pickled yam Seema Bhanwar, Arashdeep Singh, and Abhijit Ganguli Department of Biotechnology & Environmental Sciences, Thapar University, Patiala, Punjab, India Abstract The aim of this study was to characterize potential probiotic strain co-producing a-amylase and b-galactosidase. Sixty-three strains, isolated from pickle samples were screened for their hydrolase producing capacity by utilizing different starches as carbon source. One out of 63 strains, isolated from traditionally fermented pickled yam showing maximum hydrolase activity (a-amylase (36.9 U/ml) and b-galactosidase (42.6 U/ml)) within a period of 48 hours was identified as Lactococcus lactis subsp. lactis. Further, it was assessed for the probiotic characteristics under gastrointestinal conditions like acidic, alkaline, proteolytic enzymes, bile stress and found to exhibit tolerance to these stresses. The therapeutic potential of the isolate is implicated because of its antagonistic effect against enteric foodborne pathogens (Salmonella typhimurium, Escherichia coli 0157:H7, Staphylococcus aureus, Yersinia enterocolitica and Aeromonas hydrophila). The results of this study entail a potential applicability of the isolate in developing future probiotic foods besides the production of industrially significant hydrolases. Keywords a-amylase, b-galactosidase, co-production, hydrolase, Lactococcus lactis, pickled yam, probiotic History Received 29 May 2013 Revised 27 July 2013 Accepted 2 August 2013 Published online 10 September 2013 Introduction Traditional fermented foods prepared from the most common types of vegetables (such as yam, cabbage, cauliflower, turnip) are well known in many parts of the world. Some are utilized as colorants, spices, beverages and breakfast or light meal foods, while a few of them are used as main foods in the diet (Marshall & Danilo, 2011; Tamang, 2010). Pickled fruits, vegetable shoots as well as beverages prepared from cereals and edible flowers by fermentation generally form an essential constituent of the Indian population diets. These foods in the diet beyond meeting nutritional needs may modulate various physiological functions and play detrimental or beneficial roles in some diseases because of microorganisms associated with them (Kumar et al., 2012). Currently there are hundreds of traditionally fermented foods with different base materials and preparation technologies but only limited knowledge has been obtained regarding the microbiota associated with these products (Jeyaram et al., 2009). Lactic acid bacteria (LAB) are a heterogeneous group of bacteria that play a key role in the production of fermented foods and beverages with high relevance for human and animal health. These bacteria have also been recently exploited for other beneficial attributes like probiotic characteristics, bacteriocin and hydrolases production (Ruethaiwan et al., 2012). The essential characteristics for LAB to be used as probiotics during manufacturing include the following: (i) recognition as safe (GRAS; generally recognized as safe); (ii) viability during processing and storage; (iii) antagonistic effect against pathogens; (iv) tolerance to bile acid challenge; and (v) adherence to the intestinal epithelium of the host among others (Begley et al., 2005; Lin et al., 2006; MacFarlane & Cummings, 2002; Vesterlund et al., 2005). It is generally considered that minimum numbers required for a probiotic to provide a health benefit are 107 CFU/ml (Jayamanne & Adams, 2006; Ross et al., 2005). Many different microorganisms have been employed for the production of industrially important enzymes over decades. Among the group of hydrolases, two enzymes namely a-amylase and b-galactosidase are significantly important enzymes with numerous industrial applications (Konsoula & Liakopoulou- Kyriakides, 2007). a-Amylase, which catalyzes the hydrolysis of starch to low molecular weight products, is produced by a wide variety of microorganisms, but for commercial applications a-amylase is mainly derived from the microorganisms belonging to genus Bacillus (Pandey & Nigam, 2000; Violet & Meunier, 1989). For instance, a-amylases produced from Bacillus licheni- formis, Bacillus stearothermophilus and Bacillus amyloliquefa- ciens find potential application in a number of industrial processes such as in food, fermentation, textiles and paper industries (Machius & Wiegand, 1995; Pandey & Nigam, 2000). b-Galactosidase (or lactase) which hydrolyzes the milk sugar, lactose, to its components glucose and galactose, is used to treat lactose intolerant patients, prevent lactose crystallization in frozen and condensed milk products, reduce water pollution caused by whey and also to increase the sweetening properties of lactose (Furlan & Schneider, 2000; Patel & Mackenzie, 1985). In view of the advantages offered by the application of LAB to different food substrates, there is always a requirement to isolate a potential strain(s) which can be industrially important and can impart health benefits to consumers. Correspondence: Abhijit Ganguli, Department of Biotechnology & Environmental Sciences, Thapar University, Patiala, Punjab 147004, India. Tel: +91-175-2393449; Mob: 9814948811. E-mail: aganguli@ IntJFoodSciNutrDownloadedfrominformahealthcare.comby14.139.242.99on09/11/13 Forpersonaluseonly.
  • 2. In previous studies, the co-production of a-amylase and b-galactosidase from Bacillus subtilis and its employment for the hydrolysis of various organic starches (Konsoula & Liakopoulou-Kyriakides, 2007) have been reported. This paper describes the screening of various isolates from pickled vege- tables for co-production of a-amylase and b-galactosidase and further elucidates probiotic characteristics of the isolates for use as a potential probiotic strain and in other industrially important applications. The selected probiotic LAB was identified by 16S rDNA sequencing. Materials and methods Isolation of bacteria Lactic acid bacterial strains were isolated from traditionally fermented pickles procured from local markets of Punjab and Orissa, India. All samples were collected in presterile poly-bags and screw capped bottles, kept in an icebox and transported to the laboratory for analysis. Samples were plated onto MRS agar to enumerate bacteria and predominant colonies were isolated. Plates were incubated for 48 hours at 37 C. Screening of hydrolase producing lactic acid bacteria Two-stage enzymatic screening was done for a-amylase and b-galactosidase producing strains. All isolates were subjected to primary screening, while secondary screening was performed for the bacterial isolates showing enzymatic activities during the primary screening. Primary screening The starch utilizing strain was checked on modified MRS medium with RBB-soluble starch as the C-source and incubated at 37 C for 24 hours. The starch utilization was monitored by the disappearance of the blue color of the medium based on amylase production. The screened isolates were further monitored for its b-galactosidase producing potential on modified MRS agar with 30 mg/ml of 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside (X-gal) and 1% soluble starch. The production of b-galactosidase was observed after incubating the LAB inoculated MRS agar containing X-gal plate for 24 hours at 37 C. Secondary screening The screened isolates were further screened for maximum hydrolase producing potential on modified MRS agar having 1% variable starch sources, i.e. maize, potato, tapioca, fox nut and soluble starch. The enzymatic activity were determined (extra- cellulary and intracellularly) as explained by Giraud et al. (1993) and Karasova et al. (2002) for a-amylase and b-galactosidase, respectively. Enzyme isolation Cells were removed from the modified MRS medium containing 1% variable starch source (maize, potato, tapioca, fox nut and soluble starch) by centrifugation at 4500 Â g for 20 min at 4 C. The cell pellet was washed with phosphate buffer saline (PBS, pH 7.0) twice and resuspended in PBS till further use. The cell-free culture supernatant was precipitated with (NH4)2SO4 (80% saturation) followed by centrifugation (at 4500 Â g for 45 min). The precipitate was collected and resuspended in 0.1 M sodium phosphate buffer (pH 7.0) to obtain partially purified crude enzyme preparation. Protein was estimated according to the method of Bradford (1976) using crystalline bovine serum albumin as standard. Enzyme activity assay The characterization of the a-amylase producing strain was performed according to the methods of Giraud et al. (1993). The reduction in the color of the blue-colored starch–iodine complex was determined and the release of reducing sugar equivalents was estimated by the method of Miller (1959). Later, the isolates were subjected to the method of Miller (1998) to identify the strains producing b-galactosidase. Phenotypic characterization One isolate capable of hydrolyzing starch maximally was identified by its colony morphology, gram-staining and catalase test. The carbohydrate fermentation profile of the isolates was carried out by using API 50 CH strips and API 50 CHL medium according to the manufacturer’s instruction (API System; BioMerieux, Marcy I’Etoile, France). Sediment from centrifuged culture broth was used to prepare the suspension at 105 CFU/ml. Following inoculation, cultures were incubated for 4 hours at 37 C. Genotypic characterization Genomic DNA of the strain was isolated by using QIAamp DNA Mini kit as per the manufacturer’s instructions (Qiagen, Valencia, CA). Bacteria-specific universal primers used for amplification of 16S rRNA gene were the forward primer 27F (50 - AGAGTTTGATCATGGCTC-30 ) and the reverse primer 1327R (50 -CTAGCGATTCCGACTTCA-30 ) (Weisburg et al., 1991). The 16S rRNA gene was amplified in 35 cycles with a Gene Amp PCR System 2400 (Perkin Elmer, Waltham, MA). The thermal program consisted of one cycle at 94 C for 4 min, 35 cycles at 94 C for 40 s, 46 C for 40 s, 72 C for 2 min, final one cycle of 72 C for 15 s and stored at 4 C. A 100-bp DNA ladder was used as the molecular marker (Fermentas, Germany). PCR products were purified using a QIA quick PCR purification kit (Qiagen, Valencia, CA) and sequenced from both ends with an ABI3700 DNA sequencer (Applied Biosystems, Foster City, CA) using the same oligonucleotide primers used for PCR. The sequenced 16S rDNA sequences for the bacterial isolate were analyzed to detect the presence of possible chimeric artefacts and compared with the similar gene sequences using the NCBI BLAST (National Library of Medicine). The phylogenetic tree of the sequence so obtained was constructed using MEGA 4.0 software (Tamura et al., 2007). Probiotic characterization Tolerance of artificial gastric juice The ability of selected isolate to survive under gastric conditions was examined according to Casey et al. (2004) with little modifications. Overnight culture was washed with phosphate- buffered saline (PBS, pH 7.0), resuspended in synthetic gastric juice (pH 1.85 adjusted using HCl) and incubated at 37 C for 3 hours. The artificial gastric juice consisted of (g/l): 3.5 D- glucose 1.28 NaCl, 0.6 KH2PO4, 0.11 CaCl2, 0.239 KCl, 0.2 Ox bile, 0.1 lysozyme and 0.013 pepsin. Samples were withdrawn at regular intervals of 2 hours, serially diluted in PBS and enumerated on MRS agar to enumerate the viable cells. Microbial adhesion to solvents Microbial adhesion to solvents (MATS) was measured according to the method originally proposed by Rosenberg et al. (1980) and recently modified by Bellon-Fontaine et al. (1996). Briefly, bacteria were harvested in the stationary phase by centrifugation at 5000 Â g for 10 min, followed by washing in PBS (pH 6.2) twice and resuspending in 0.1 M PBS (pH 6.2) to an optical 2 S. Bhanwar et al. Int J Food Sci Nutr, Early Online: 1–9 IntJFoodSciNutrDownloadedfrominformahealthcare.comby14.139.242.99on09/11/13 Forpersonaluseonly.
  • 3. density of 0.4 at 600 nm (A0) (approximately 108 CFU/ml cell density). Further, 0.2 ml of solvent was added to 1.2 ml of cell suspension. Following pre-incubation at room temperature for 10 min, the two-phase system was mixed on a vortex for 2 min. To allow complete phase separation of the mixture, the aqueous phase was removed after 15 min and its optical density at 600 nm (A1) was measured. The percentage of microbial adhesion to solvent was calculated as 1 À ðA1=A0Þ½ Š à 100: n-Hexadecane (polar), Chloroform (a monopolar and acidic) and ethyl acetate (a monopolar and basic) were used as solvents in this study. Resistance to 0.4% (v/v) phenol The ability of selected LAB strain to grow in the presence of phenol was tested by inoculating cultures (1% of overnight culture) in MRS broth with and without 0.4% phenol. Serial dilutions were spread-plated (100 ml aliquots) onto MRS agar at time 0 and after 24 hours of incubation at 37 C to enumerate surviving bacteria (Xanthopoulos et al., 1999). Bile salt hydrolase activity Bile salt hydrolase (Bsh) activity of the culture was detected using the plate screening procedure described by Du Toit et al. (1998). Briefly, overnight culture was spotted onto MRS agar plates containing 0.5% (w/v) sodium taurodeoxycholate (Sigma, St. Louis, MO) and 0.37 g/l CaCl2. Colonies with precipitation zones were considered Bsh-positive. Lactobacillus acidophilus ATCC 43121 and E. faecium E 179 were used as Bsh-positive and Bsh- negative control strains, respectively. Determination of antibiotic resistance The selected strains were investigated for their antibiotic resistance profile using the E-test (Viva Diagnostika, Cologne, Germany) using MRS agar and anaerobic incubation conditions and following the manufacturer’s instructions. The minimum inhibitory concen- tration (MIC) values used to determine whether strains were susceptible or resistant were those as suggested by Scientific Committee for Animal Nutrition (SCAN) (Chesson et al., 2002). Determination of antimicrobial potential of probiotic strains Screening for antagonistic activity: The agar spot test as described by Schillinger Lucke (1987) was used for screening for antagonistic activity of the selected strains against a variety of indicator strains: Staphylococcus aureus ATCC 9144, Aeromonas hydrophila ATCC 35654, Yersinia enterolitica MTCC 840, Salmonella typhimurium ATCC 19585 and Escherichia coli 057:H7 ATCC 43895. The agar spot test method of (Uhlman et al., 1992) was further used to test the activity of cell-free neutralized supernatants. The supernatants were tested against the same indicator strains used above. Production of H2O2: Overnight cultures (10 ml) were spotted onto ABTS (2-20 -azino-di-3-ethylbenzthiazoline-6-sulfonic acid)- agar plates as described by Kostinek et al. (2005). The plates were incubated anaerobically at 37 C for 72 hours and then were exposed to the atmosphere and H2O2 producing strains were considered positive when a violet halo surrounded the colony appeared (Marshall, 1979). Statistical analysis All the experiments were performed in triplicate. Error bars on graphs show the standard deviation. The data were analyzed by the analysis of variance (ANOVA) and the means of enzyme activity were compared by Tukey’s test using GraphPad Prism version 5.0 (GraphPad Prism Software, Inc., La Jolla, CA). Results Isolation and primary screening of hydrolase producing LAB Of the 63 indigenously isolated bacterial strains screened for a-amylase and b-galactosidase production, 20 showed positive result. Bacterial strains showed the formation of white colored colonies on RBB starch agar medium indicating amylolytic activity whereas blue colur colonies on X-gal agar medium indicating b-galactosidase activity (data not shown). Out of 20 isolates, only six isolates namely LA-1, LA-2, LA-3, LA-4, LA-5 and LA-6 showed high b-galactosidase and amylolytic activity (Table 1) and were subjected to further screening using different variable starch sources. Secondary screening The co-production of a-amylase and b-galactosidase by screened isolates was investigated in order to elucidate the source (intracellular/extracellular) of enzyme production. Maximum a-amylase activity was detected in the supernatant in comparison to whole cells, the isolate suggesting the enzyme to be extracel- lular whereas in case of b-galactosidase, maximum activity was exhibited by cell-free extracts in comparison to supernatant signifying an intracellular location of b-galactosidase. The isolate LA-6 exhibiting maximum enzyme activity (Figure 1) was selected to investigate the effect of different organic substrates on the co-production of a-amylase and b-galactosidase. Effect of various organic substrates on a-amylase and b-galactosidase co-production Initially bacterial growth was carried out at 37 C with agitation at 120 rpm and pH was maintained at 6.5. A modification of MRS medium was adopted with 20 g/l of starches (maize, potato, tapioca, fox nut and soluble starch) replacing glucose. Samples were withdrawn at regular intervals of 2 h and analyzed for enzyme activity. Figure 2 shows the production of a-amylase and b-galactosidase in the presence of various starches as carbon sources at 2% (w/v) concentration. Although all carbon sources supported good growth of the microorganism, soluble sugars were easily metabolized carbon sources giving relatively higher growth in comparison to starch. Enzyme production Maximum a-amylase activity was detected in the supernatant in comparison to whole cells (Figure 1), suggesting the enzyme to be extracellular whereas in case of b-galactosidase, maximum activity was exhibited by cell-free extracts in comparison to supernatant (Figure 1) signifying an intracellular location of b-galactosidase. Table 1. Screening of lactic acid bacteria for a-amylase and b-galactosidase activity. Bacteria a-Amylase activity (U/ml) b-Galactosidase activity (U/ml) LA-1 22.1 21.7 LA-2 24.6 23.3 LA-3 19.4 20.5 LA-4 27.3 22.8 LA-5 19.9 27.1 LA-6 36.9 42.6 DOI: 10.3109/09637486.2013.832175 Probiotic characterization of Lactococcus lactis 3 IntJFoodSciNutrDownloadedfrominformahealthcare.comby14.139.242.99on09/11/13 Forpersonaluseonly.
  • 4. Kinetics of a-amylase and b-galactosidase production The isolate was grown in potato starch exhibited b-galactosidase activity after 4 hours of incubation. The activity increased gradually with incubation time and reached highest at 14–16 hours of cellular growth (Figure 3). The total a-amylase activity initially increased and was maximum following 12–14 hours of incubation. This initial part where the increase in activity was observed comprised the exponential phase and early stationary phase. However, as incubation continued, total a-amylase activity became stable from 40–48 hours and decreased further. In general, a decline in total enzyme activity in both cases was observed after the stationary phase during the growth experi- ments. The decline in total enzyme activity could be attributed to inhibition of cellular functions due to lowering of pH, depletion of nutritional factors from the growth medium, deactivation of the enzyme due to low pH catabolite repression, or/and inducer exclusion. LAB maintains a cytoplasm that is more alkaline than the medium, but the medium is acidified during growth by secretion of lactic acid. However, if the cytoplasmic pH decreases below a threshold pH, cellular functions are inhibited and the intracellular enzymes can be deactivated (Kashket, 1987). Phenotypic characterization The isolate LA-6 capable of hydrolyzing starch maximally was investi
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