Use of Lactobacilli in cereal-legume fermentation and as potential probiotics towards phytate hydrolysis
Authors: Amritha Girish K 1 and Venkateswaran G1*
1Microbiology and Fermentation Technology Department, CSIR- Central Food Technological Research Institute, Mysuru, Karnataka, India
*Corresponding author:
Venkateswaran G
Chief Scientist and Head
Microbiology and Fermentation Technology
CSIR-Central Food Technological Research Institute
Mysuru-570 020, India
Phone: 0821-2517539
Email: /
Acknowledgements Authors thank the Director, CSIR-CFTRI for providing necessary research facilities. Amritha Girish K is grateful to Council of Scientific and Industrial Research (CSIR), New Delhi for the fellowship grant. Authors also acknowledge Dr. Prakash. M. Halami for his kind support.
Abstract
Phytate is a potent inhibitor of mineral absorption in humans occurring in plant-based food. Application of lactobacilli that produce phytate-degrading enzymes (phytases) to reduce phytate is an interesting, yet a not much explored sector of research. Therefore, phytate dephosphorylation by Lactobacillus plantarum MTCC 1325 was evaluated. Cells at stationary phase showed phytase activity which was maximal at 24 h of growth. Glucose concentration and the type of phosphorous source in the media modulated the enzyme activity. Fermentation of cereal and/or legume flours with the strain resulted in phytate reduction with the highest in sorghum (73%) and the lowest in horse gram (34%). Further, the strain showed tolerance to acid, bile and simulated gastrointestinal fluid. Significant phytase activity in the presence of simulated gastrointestinal fluids along with the ability to produce phytases post exposure to simulated gastrointestinal fluids is of interest. To the best of our knowledge, this is the first report on the effect of simulated gastrointestinal fluid on cell-associated phytases of lactobacilli. The results of the investigation indicate that Lactobacillus plantarum MTCC 1325 could be used as a starter in cereal-legume fermentation and as potential probiotics to achieve phytate hydrolysis in food matrices and also in gastrointestinal tract.
Key words Phytate. Lactobacillus plantarum. phytate-degrading enzyme. cereal-legume fermentation. Probiotics. simulated gastrointestinal conditions
Introduction
Phytic acid (myo-inositol hexakisphosphate or phytate in salt form) is the principal storage form of phosphorus in many plant tissues [1]. At physiological pH, the negatively charged phosphate groups of phytate bind to di and trivalent cations and proteins. These complexes are unabsorbable by the human gastrointestinal tract due to its limited ability to produce phytate-degrading enzymes (phytases) [2]. Hence, the molecule is considered an antinutrient. Phytate dephosphorylation is of nutritional importance as it results in increased bioavailability of essential dietary components [3].
Phytases have been detected in several microorganisms including Lactic Acid Bacteria (LAB) [4, 5]. Lactic Acid Bacteria are a group of organisms that impose health benefits by live colonization of host cells [6]. They are Generally Regarded as Safe (GRAS) as they do not pose any health risk to the host [7]. Additionally, they produce enzymes involved in food processing [8]. Therefore, LAB are attractive candidates with respect to phytate reduction due to their role in food fermentations and also as probiotics [9].
Phytase activity is a desirable technological trait in LAB intended to be used as starters in cereal and legume fermentations. Whole wheat bread and soy drinks with reduced phytate content have been formulated by utilizing LAB with phytate hydrolyzing potential as starters [10, 11]. An entirely different approach to bring about phytate hydrolysis in the human gastrointestinal tract consists of administering probiotics with phytase activity [12]. Reduction in the anti nutritive effects of phytate was reported when livestock animals were fed with recombinant LAB expressing phytase gene [13]. However, phytases from probiotics should retain its activity under gastrointestinal conditions [14].
The aim of the present investigation was to evaluate phytate degradation by Lactobacillus plantarum MTCC 1325. The ability of the strain to degrade phytate from natural sources and also the effect of simulated gastrointestinal fluids on its phytase activity was determined. Thus, the potential technological role of L. plantarum MTCC 1325 to reduce phytate during food fermentation and in the gastrointestinal tract is discussed.
Materials and methods
Materials
Microbiological media components were from Hi Media (Mumbai, India). Sodium phytate and taurodeoxycholate sodium salt (TDCA) were obtained from Sigma (St Louis, MO, USA). All other chemicals of analytical grade were purchased from Sisco Research Laboratories (India). Finger millet (Eleusine coracana), Sorghum (Sorghum biclor), Wheat (Triticum aestivum), Black-eyed beans (Vigna ungiculata), Green gram (Vigna radiata) and Horse gram (Macrotyloma uniflorum) were procured from the local market (Mysuru, Karnataka, India). Cereals and pulses were cleaned and milled to fine flour in a mixer grinder and stored at 4 ºC.
Bacteria and culture conditions
Lactobacillus plantarum MTCC 1325 was procured from Microbial Type Culture Collection, Chandigarh, India. The strain was maintained at -20 ºC in MRS (De Man, Ragosa and Sharpe) broth with 40% glycerol (v/v). The culture was activated from glycerol stock by propagating in MRS broth twice.
For phytate-degradation studies, the strain was grown in modified MRS (MRS-MOPS, pH 6.5±0.2). The media was prepared by replacing KH2PO4 with 0.65 g/L sodium phytate and 0.1 M N-(Morpholino)-propanesulfonic acid (MOPS). Also, beef and yeast extracts were reduced to 4 and 2 g/L respectively [15]. The media was inoculated with 1% (v/v) overnight grown culture propagated under similar conditions and incubated until the stationary phase at 37 ºC. The cells were harvested (7000 g, 10 min, 4 ºC), washed with 0.05 M Tris-HCl (pH 6.5) and resuspended in 0.1 M sodium acetate buffer (SAB) of pH 5.5 [16]. Subsequently, the cell suspension was used to determine phytase activity.
To determine the optimal cultivation time for maximum phytase activity, cells were grown in MRS-MOPS as described earlier. Growth was monitored by measuring the absorbance at 600 nm (UV-Vis Spectrophotometer, Shimadzu, Japan). At every 6 h intervals, an aliquot of the culture broth was withdrawn to estimate the phytase activity of the cells.
Enzyme production was also studied in MRS-MOPS with different concentrations of glucose (0.5, 1, 2, 4 g/L), MRS with sodium phytate (0.65 g/L) and standard MRS media. Bacterial cell counts were obtained by pour plating on MRS agar followed by incubation at 37 ºC for 24 h.
Phytase activity assay
Phytase activity assay was performed using sodium phytate as the substrate. The reaction mixture consisted of 400 µL of 0.1 M SAB (pH 5.5) containing 2 mM sodium phytate and 200 µL enzyme. After incubation at 50 ºC for 40 min, the reaction was terminated by the addition 100 µL of 2% (w/v) trichloroacetic acid (TCA) [16]. An aliquot of the reaction mixture was used to determine the liberated inorganic phosphate (Pi) [17]. Controls were run by addition of enzyme after TCA. A unit (U) of phytase is defined as 1.0 µmole of Pi liberated per min under assay conditions. The protein in the samples was estimated [18] and enzyme activity was expressed as specific activity (U/mg protein).
Fermentation of cereals and legumes
Cereal and legume flours were autoclaved (15 psi, 121 ºC, 30 min) and checked for residual phytase activity. Fermentation of the autoclaved flours with the test strain was carried out according to Reale et al [19] with slight modifications. Briefly, 10 g of flour was suspended in 100 mL sterile water under aseptic conditions. The mixture was inoculated at 1% level with saline suspension (108 CFU/mL) of the test strain. The producer strain grown in MRS for 16-18 h was harvested (7000 g, 10 min, 4 ºC) and suspended in equal volume of saline to obtain bacterial cell suspension. The inoculated flour samples were incubated at 37 ºC at 120 rpm for 24 h in shaker incubator (MaxQ 4450, Thermo Scientific, Bangalore, India). Controls consisted of cereal-legume suspension without inoculation.
Phytic acid extraction and estimation
The fermented cereal or legume mixture was freeze-dried (Freeze zone freeze dry system, Labconco, USA) and subsequently used for phytate extraction and estimation [20]. Sodium phytate was used as the standard.
Acid, Bile tolerance and Bile salt hydrolase (BSH) activity of L. plantarum MTCC 1325
Tolerance to acid (pH 3.0) and bile salts (0.3% w/v of ox-bile) was performed as described by Archer and Halami [21]. BSH activity was detected on MRS agar plates supplemented with 0.5 % (w/v) TDCA and 0.37 g/L CaCl2 [22].
Survival of L. plantarum MTCC 1325 in simulated gastrointestinal fluids
Simulated gastric fluids (pH 2.5) was prepared according to Zarate et al [23] while simulated intestinal fluids (pH 8.0) was formulated as described by Bao et al [24]. Survival of L. plantarum MTCC 1325 in simulated gastrointestinal fluid was carried out according to Archer and Halami [22] with modifications. Briefly, overnight grown culture was inoculated into simulated gastric fluids at 5% level and incubated at 37 ºC in a shaker incubator at 200 rpm. Viable cell counts were obtained at 0, 90, and 180 min of incubation to determine bacterial survival in gastric fluid. Similarly, simulated intestinal fluids were inoculated and incubated as described above. To determine bacterial survival, viable cell counts were determined at 0, 3, 6 and 24 h of incubation. Percentage survival was calculated using the formula:
% survival = log CFU N1/ log CFU N0 x100
Where N1 is the viable count after exposure to simulated gastrointestinal fluids for specific time intervals and N0 represents cell count before treatment.
Cell surface hydrophobicity of L. plantarum MTCC 1325
Cell surface hydrophobicity was determined using xylene and hexadecane as described earlier [25]. Briefly, L. plantarum cultured in MRS for 16-18 h at 37 ºC were harvested (7000 g, 4 ºC, 10 min) and washed twice with phosphate urea magnesium (PUM) buffer. The cells were resuspended in PUM buffer to an absorbance of ~0.7 (initial OD) at 600 nm. Bacterial cell suspension (3 mL) was mixed with 1 mL hydrocarbon (xylene or hexadecane) and vortexed thoroughly. The mixture was incubated at 37 ºC for 1 h for phase separation. The absorbance of the aqueous phase was taken, and percentage surface hydrophobicity was calculated as follows:
% surface hydrophobicity = (A0−A1) /A0 X 100
Where A0 is the intial and A1 is the final OD
Phytase activity of L. plantarum MTCC 1325 under simulated gastrointestinal conditions
Initially, the phytase activity of L. plantarum MTCC 1325 was determined in the presence of simulated gastrointestinal fluids as described by Gonzalez-Cordava et al [26] with minor modification. Simulated gastrointestinal fluids were prepared as described earlier. Equal volume of bacterial cell suspensions and simulated gastric or intestinal fluid was incubated for 0, 15, 30 and 60 min at 37 ºC. After incubation sodium phytate was added to the reaction mixture to assay phytase activity as described earlier.
Additionally, the potential of L. plantarum MTCC 1325 to produce phytases after exposure to simulated gastrointestinal fluids was determined based on the procedure by Gonzalez-Cordova et al [26].
Active cultures of L. plantarum in MRS (5 mL) were harvested (7000 g, 10 min, 4 ºC) and washed twice with 0.05 M Tris-HCl (pH 6.5). The cells were resuspended in equal volume of simulated gastric or intestinal fluid (~108 CFU /mL) and incubated at 37 ºC for 0, 180 and 240 min in a shaker incubator. Post incubation, cells were harvested, washed as described earlier and resuspended in 4 mL of Tris-HCl. Further, MRS-MOPS broth was inoculated at 1% with the cell suspension and incubated at 37 ºC for 24 h. Following incubation, the cells were processed and assayed for phytase activity.
Statistical analysis
The results obtained are mean of three independent determinations ± standard deviation (SD). Multiple sample comparisons were statistically analyzed with SPSS16 [27]. Tukey’s least significance difference (LSD) test was used to compare means at 5% significance level.
Results and discussion
Phytate degradation by L. plantarum MTCC 1325
Screening of LAB from various sources including culture collection centers for enzymatic phytate-degradation showed L. plantarum MTCC 1325 to be a promising strain [28]. The activity was exclusively cell-associated in accordance to previous studies [4, 5, 16]. Production of phytate-degrading enzyme by the culture in MRS-MOPS media started with the onset of stationary phase and reached a maximum at 24 h (Fig. 1). Most phytate-degrading LAB showed peak activity in the stationary phase [4, 29].
Glucose concentration and phosphorous source are known to modulate microbial phytase production [30]. The effects of glucose concentration and phosphorus source on the phytase activity of L. plantarum MTCC 1325 are presented in Table 1. Increased activity, with no significant difference in cell biomass (p 0.05) was observed when media glucose was augmented to 1% from 0.5%. Activity slightly decreased with 2% glucose, with growth yields showing no significant difference. Phytase activity with 4% glucose was comparable to the media with 2% glucose; however there was slight decrease in growth. Lactobacillus amylovorus produced maximum phytase at 1% glucose [31].
Cells grown in MRS with KH2PO4 as phosphorous source showed the least phytate-degrading ability. Activity improved on supplementing phytate to MRS. Cells cultivated in MRS-MOPS based media with phytate as sole source of phosphorous showed the highest phytase activity. The results indicate the stimulatory effect of phytate on enzyme production. Similar trend was observed in other LAB strains that produced phytate-degrading enzymes [16, 29]. Growth yields were highest in the nutritionally rich MRS media and lowest in the minimal MRS-MOPS media.
Fermentative reduction of phytate in cereal and legume flours by L. plantarum MTCC 1325
Fermentation of cereal and legumes with L. plantarum MTCC 1325 resulted in considerable dephytinization of the substrates (Table 2). The substrates also supported the growth of the bacterium (online resource 1). However, reduction rates varied with the highest in sorghum (73.6%) and the lowest in horse gram (34.6%). No significant reduction was observed with green gram as the substrate (p < 0.05). Similarly, of various substrates tested, only wheat flour showed phytate reduction when phytate hydrolyzing strain of Lueconostoc mesenteroides was used as starter [32].
Autoclaving the cereal and legume flours prior fermentation ensured that the observed reduction in phytate was due to microbial activity. Similarly, autoclaved finger millet flour was dephytinized with Lactobacillus pentosus CFR3 which showed phytase activity [28]. Soy curd with reduced phytate was also obtained on fermentation of autoclaved soy milk with L. mesenteroides [33].