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Dephytinization of seed coat matter of finger millet (Eleusine coracana) by Lactobacillus pentosus CFR3 to improve zinc bioavailability
Accepted Manuscript Dephytinization of seed coat matter of finger millet (Eleusine coracana) by Lactobacillus pentosus CFR3 to improve zinc bioavailability Girish K. Amritha, Usha Dharmaraj, Prakash M. Halami, G. Venkateswaran PII:
S0023-6438(17)30699-0
DOI:
10.1016/j.lwt.2017.09.024
Reference:
YFSTL 6539
To appear in:
LWT - Food Science and Technology
Received Date: 25 May 2017 Revised Date:
24 August 2017
Accepted Date: 15 September 2017
Please cite this article as: Amritha, G.K., Dharmaraj, U., Halami, P.M., Venkateswaran, G., Dephytinization of seed coat matter of finger millet (Eleusine coracana) by Lactobacillus pentosus CFR3 to improve zinc bioavailability, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.09.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dephytinization of seed coat matter of Finger Millet (Eleusine coracana) by
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Lactobacillus pentosus CFR3 to improve Zinc bioavailability
3 Authors: Amritha Girish K1, Usha Dharmaraj2, Prakash M. Halami1, and G.
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Venkateswaran1*
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Microbiology and Fermentation Technology Department
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Grain Science Technology Department
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CSIR-Central Food Technological Research Institute
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Mysuru-570 020
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Karnataka, India
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G. Venkateswaran
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Chief Scientist & Head
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Microbiology and Fermentation Technology
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CSIR- Central Food Technological Research Institute
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Mysuru-570 020
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Karnataka, India
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Email: [email protected] /[email protected]
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Abstract
25 Finger millet seed coat matter (SCM) is a rich source of dietary fiber, calcium, iron and
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zinc (Zn). However, the bioavailability of minerals especially Zn is limited as it occurs as
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insoluble complexes with phytate. This study envisages the potential of phytase-active
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Lactobacillus pentosus CFR3 to dephytinize SCM derived from native (non-processed),
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malted and hydrothermally treated finger millets with an expectation to improve its Zn
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bioavailability. After 24 h fermentation, the phytate in native, malted and hydrothermally
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treated SCM reduced to 56.70%, 66.65% and 87.85% respectively. Correspondingly,
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Phytate/Zn molar ratios decreased to 18.20, 19.20 and 22.32 indicating improved Zn
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bioavailability. Also, Zn bioacessiblity from native, malted and hydrothermally treated
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SCM increased to 28.40%, 34.57% and 12.10 % as determined by in vitro dialyzability
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experiments following 24 h fermentation. Therefore, fermentation of SCM with L.
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pentosus CFR3 represents a safe and feasible solution to counteract the effects of phytate
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on Zn absorption.
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Keywords: Seed coat; finger millet; Lactobacillus pentosus; dephytinize; Zinc
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1. Introduction
48 The seed coat matter (SCM) from native, malted and hydrothermally treated finger millet
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is a byproduct of the millet processing industry. It contains significant proportion of
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minerals, dietary fiber and phytochemicals. In the recent years, SCM has been utilized as
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adjuncts in cereal foods to obtain products with high levels of dietary fiber, calcium (Ca),
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iron (Fe) and zinc (Zn) (Krishnan, Dharmaraj, & Malleshi, 2012; Krishnan, Dharmaraj,
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Manohar, & Malleshi, 2011). However, the bioavailability of minerals from SCM may be
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limited due to the presence of phytate (myo-inositol hexakisphosphate) (Poutanen,
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Flander, & Katina, 2009). The phosphate groups of phytate form stable complexes with
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dietary cations, thus hindering their bioavailability (Liang, Han, Nout, & Hamer, 2008).
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Zinc bioavailability is most severely affected by dietary phytate (Lönnerdal, 2002;
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Lopez, Leenhardt, Coudray, & Remesy, 2002). Several studies have revealed a positive
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correlation between phytate-rich diets and Zn deficiency in humans (Lönnerdal, 2002).
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Clinical manifestations of Zn deficiency include growth retardation, hypogonadism in
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males, rough skin, impaired immunity, neuro-sensory disorder and cognitive impairment
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(Lönnerdal, 2002; Prasad, 2012). Therefore, optimization of Zn bioavailability is of
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significance.
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Enzymatic phytate hydrolysis is a promising strategy to improve Zn absorption in
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humans (Fischer, Egli, Aeberli, Hurrell, & Meile, 2014). Food ingredients have been
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successfully dephytinized with phytate-degrading enzymes (phytases) (Haros, Rosell, &
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Benedito, 2001; Marklinder, Larsson, Fredlund, & Sandberg, 1995; Sandberg, Hulthen,
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& Turk, 1996). Several researchers have documented the presence of phytases in Lactic
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Acid Bacteria (LAB) (De Angelis et al., 2003; Palacios, Haros, Rosell, & Sanz, 2005;
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Raghavendra & Halami, 2009; Zamudio, González, & Medina, 2001). This opens up
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possibilities towards fermentative reduction of phytate with specific phytase-active LAB.
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The application of LAB to dephytinize food ingredients is safe due to the GRAS
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(Generally Regarded as Safe) status conferred on this bacterial group. Moreover, it is
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cheaper compared to the use of purified phytase.
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Elimination of phytate from byproducts of millet milling with specific LAB is a
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relatively unexplored area of research. Therefore, the current investigation focused on the
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dephytinization of SCM from native, malted and hydrothermally treated finger millets by
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Lactobacillus pentosus CFR3 as a means to improve Zn bioacessibility.
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2. Materials and methods
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2.1. Sample preparation
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Finger millet (GPU 28 Variety) was procured from University of Agriculture Sciences,
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Bangalore, Karnataka, India. Native SCM (NSCM) was prepared as described by
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Chethan and Malleshi (2007). Malted SCM (MSCM) was obtained following the protocol
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given by Malleshi and Desikachar (1979), while hydrothermally treated SCM (HTSCM)
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was prepared as described by Krishnan et al. (2012). The SCM samples were finely
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powdered in a laboratory grinder, passed through a 60 BSS mesh and stored at 4 ºC until
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analysis.
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2.2. Materials
94 Dialysis membrane (10 kDa cut-off), hydrogen peroxide (H2O2; 30%), lanthanum
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chloride (LaCl3), lysozyme, mineral standards (Ca and Zn), nitric acid (HNO3; 90%),
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sodium phytate and wheat phytase were from Sigma (St Louis, MO, USA).
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Microbiological media components, pancreatin, pepsin and ox-bile were purchased from
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Hi Media (Mumbai, India). All other reagents of analytical grade were obtained from
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Sisco Research Laboratories (India). Deionized water from Millipore system and acid-
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washed glassware were used for the entire study.
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2.3. Bacterial strain
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Lactobacillus pentosus CFR3 which produced phytate-degrading enzyme was used in the
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study (Amritha, Halami, & Venkateswaran, 2017). The culture was maintained at -20 ºC
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in MRS (De Man, Ragosa and Sharpe) broth with 40% (v/v) glycerol. The strain was
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activated from the glycerol stock by subculturing in MRS broth twice.
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2.4. Preparation of crude phytase (CP) from L. pentosus CFR3
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Lactobacillus pentosus CFR3 was propagated in modified MRS broth (pH 6.5 ± 0.2)
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under static condition for 8 h at 37 ºC. Modified MRS broth was prepared by replacing
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glucose with maltose. Also, sodium di-hydrogen phosphate (KH2PO4) was replaced with
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sodium phytate (0.65 g/L) and 0.1 M 3-[N-morpholino] propanesulfonic acid (MOPS).
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The cell-associated phytase from the culture was extracted with lysozyme and NaCl
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(Amritha et al., 2017).
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2.5 Dephytinization of SCM with CP of L. pentosus CFR3
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The SCM samples (0.5 g) were suspended in 2 mL of CP (~8 mg protein) and incubated
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in a shaking water bath at 50 ºC for 120 min. Similarly, samples suspended in 0.25%
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(w/v) wheat phytase (0.01 - 0.04 U/mg) in sodium acetate buffer (0.1 M, pH 5.5) and
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sodium acetate buffer constituted the positive and negative controls respectively. Post
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incubation; samples were freeze-dried (Freeze Zone Freeze dry System, Labconco, USA)
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and subjected to phytate extraction and estimation.
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2.6. Inoculum preparation and fermentation of SCM with L. pentosus CFR3
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Lactic fermentation of SCM were carried out based on the procedure given by Reale,
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Konietzny, Coppola, Sorrentino, and Greiner (2007). Lactobacillus pentosus CFR3 was
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propagated in MRS for 16 ± 2 h at 37 ºC. Cells were harvested (7000 g, 10 min, 4 ºC),
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washed and resuspended in an equal volume of saline to obtain a cell suspension (108
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CFU/mL). The SCM samples were autoclaved (15 psi, 121 ºC, 30 min) and subsequently
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suspended in sterile water under aseptic conditions to obtain a 10% (w/v) suspension. The
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SCM-based suspensions were inoculated with saline suspension of the producer strain
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and incubated at 37 ºC in a shaker incubator (New Brunswick, USA) at 150 rpm.
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Uninoculated control was run in parallel.
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2.7. Determination of viable cell counts and pH
140 Microbial cell counts were obtained by pour plating on MRS agar. The plates were
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incubated at 37 ºC for 24-48 h. Changes in pH of the fermenting SCM was noted with a
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pH meter (Eutech Instruments, Singapore).
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2.8.1. Phytic acid
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Phytate was extracted and estimated from SCM samples as described by Gao et al. (2007)
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with sodium phytate as the standard.
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Total minerals (Ca and Zn) were estimated according to Kumari and Platel (2016) with
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minor modifications. Briefly, samples (0.1 g) were wet digested for 24 h with 0.5 mL
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HNO3 and 0.3 mL H2O2, followed by gradual refluxing in a hot plate. Subsequently, the
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volume was made up to 10 mL with 0.5% HNO3. The samples were filtered through 0.45
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µm syringe filters (Millex HV, Millipore) and appropriately diluted before estimation
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using Flame Atomic Absorption Spectrometry (FAAS; Shimadzu AAF-6701). For Ca
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estimation, the samples and standards were spiked with 0.1% (w/v) LaCl3 to prevent
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phosphorous interference.
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2.8.2. In vitro dialyzable Zn
163 In vitro dialyzable Zn was determined by equilibrium dialysis method as described by
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Luten et al. (1996) with modifications. In short, samples (10 g) were suspended in 80 mL
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water and pH was adjusted to 2.0 with 6.0 M HCl. Subsequently, pepsin solution (3 mL
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of 16 g/100 mL pepsin in 0.1 M HCl) was added to the mixture and the volume was
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raised to 100 mL. The mixture was incubated in a shaker at 37 ºC for 2 h at 110 rpm to
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obtain gastric digests. The gastric digests were frozen for 30 min to stop the reaction.
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Titratable acidity (TTA) was measured in an aliquot (20 mL) of the gastric digest
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containing 5 ml of pancreatin-bile mix (5 g pancreatin + 25 g ox-bile in 1 L of 0.1 M
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sodium bicarbonate (NaHCO3)) by adjusting the pH to 7.5 with 0.2 M sodium hydroxide
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(NaOH). An aliquot of gastric digest (20 mL) was now subjected to intestinal digestion in
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a beaker containing dialysis membrane filled with 25 mL of NaHCO3 equal in moles to
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the NaOH as determined by TTA. The beakers with the dialysis membranes were
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incubated in a shaker as described above until the pH of the digest changed to 5.0.
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Pancreatin-bile mix was added (5 mL) and the incubation was continued until the pH
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reached 7.0. After incubation, dialysis membranes were rinsed with distilled water and its
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volume was noted. Further, the dialysates (bioavailable fraction) were acidified with
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HNO3 at 5% level, centrifuged (10,000 g) and filtered before estimating the Zn content
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by AAS. Bioacessibility was calculated as follows
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Bioaccessibility (%) = 100 x Y/Z where,
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Y is the Zn content in bioavailable fraction (mg of mineral element/ 100 g sample) and Z
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is the total Zn (mg mineral element/100 g sample).
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2.9. Statistical analysis
186 Results were expressed as mean of triplicates ± standard deviation (SD). Testing for
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statistical significance was done by one-way ANOVA. Post hoc comparison was
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performed by Dunnet's and Tukey's tests. Differences were considered significant
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when p <0.05. All analyses were performed using GraphPad Prism 7 (GraphPad Software
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Inc., San Diego, CA, USA).
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3. Results and Discussion
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3.1. Dephytinization of SCM by treatment with CP
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Seed coat matter from native, malted and hydrothermally treated finger millets have been
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used as adjuncts in the preparation of cereal-based food (Krishnan et al., 2011).
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Therefore, we examined the ability of L. pentosus CFR3 to dephytinize all three types of
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SCM. To emphasize enzymatic nature of phytate reduction, NSCM, MSCM and HTSCM
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were treated with CP from the test strain. Under given conditions, phytate in NSCM,
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MSCM and HTSCM reduced to 86.75%, 89.39% and 96.03% respectively (Fig. 1). The
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observed reduction in phytate was attributed to CP as the substrates were autoclaved to
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rule out plant phytases if any. A more pronounced phytate reduction was noted when
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CP from L. pentosus CFR3 was used to dephytinize whole finger millet flour (Amritha et
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al., 2017).
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3.2. Fermentative reduction of phytate from SCM
209 Removal of phytate through fermentation is simpler and cost-effective compared to the
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use of purified phytases. Therefore, further studies were in this direction. Phytate-
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degrading LAB has been successfully grown in cereal-based substrates with concomitant
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phytate reduction (Anastasio et al., 2010; Raghavendra, Ushakumari, & Halami, 2011).
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However, there are no reports on fermentative reduction of phytate from finger millet
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SCM by L. pentosus. Further, the major nutrients in all three SCM samples were
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comparable (Krishnan et al., 2011). The initial pH of NSCM and HTSCM was similar
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(5.60-5.8) while that of MSCM was slightly lower (~4.80).
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Inoculum size standardization was carried out by fermenting SCM with varying
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levels of inoculum for 16 ± 2 h (Fig. 2). A positive correlation was observed between
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inoculum size and phytate reduction. Since reduction rates obtained with 5% and 7%
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inoculum were statistically insignificant (p <0.05), 5% inoculum was used for time
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course experiments. Lower inoculum levels may not be adequate for initiating growth
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and enzyme synthesis on different substrates. In general, increased inoculum size
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enhances microbial growth and enzyme synthesis to a certain level. A balance between
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the proliferating bacterial biomass and the fermentation substrates is critical for optimal
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enzyme activity (Sabu, Augur, Swati, & Pandey, 2006).
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Growth of L. pentosus CFR3 and evolution of pH during 24 h fermentation of
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SCM is shown in Figs. 3a and 3b. Initial phytate content of NSCM, MSCM and HTSCM
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were 580.03, 384.40 and 730.24 mg/100 g respectively. After 24 h fermentation, phytate
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levels dropped to 56.7%, 66.65% and 87.85% (Fig. 3c). Phytate reduction in NSCM was
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gradual, while phytate in MSCM decreased drastically within 3 h after which significant
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hydrolysis was not observed. In case of HTSCM, phytate reduction was less compared to
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NSCM and MSCM. The unique patterns of phytate degradation observed with the three
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SCM samples may be due to the differences in initial phytate concentration and the
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nature of phytate (Lan, Abdullah, Jalaludin, & Ho, 2002). Interestingly, HTSCM which
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did not show much phytate degradation showed a similar trend when treated with CP.
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3.3. Molar ratios of phytic acid to zinc (PA/Zn)
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Phytate hydrolysis is likely to improve Zn bioavailability as it is the single major
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inhibitor of Zn bioavailability (Lönnerdal, 2002). Phytate to zinc molar ratio is assumed
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to provide an indication of the Zn bioavailability, with PA/Zn molar ratios>15 hinting
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towards reduced Zn absorption (Liang et al., 2008; Ma et al., 2005). Although a decrease
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in PA/Zn values were observed for 24 h fermented SCM (Table 1), they were slightly
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above the critical threshold. However, an increase in Zn bioavailability is expected.
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Several researchers have reported reduced PA/Zn molar ratios in cereal and legume
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based food substrates after lactic fermentation (Fischer, Egli, Aeberli, Hurrell, & Meile,
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2014; García-Mantrana, Monedero, & Haros, 2015; Najafi, Rezaei, Safari, & Razavi,
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2012). Since finger millet SCM is very rich in Ca (Krishnan et al., 2012), PA/Ca ratios
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were also calculated. Calcium bioavailability is compromised when PA/Ca exceeds 0.24
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(García-Mantrana et al., 2015; Ma et al., 2005). In this context, calcium bioavailability
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from none of the samples is likely to be impaired.
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3.4. In vitro dialyzability of Zinc
255 Ideally bioavailability studies should be carried out in vivo in human subjects because
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data from animal models are questionable due to differences in the physiology between
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humans and model organism. However, human trials are expensive and difficult (Amalraj
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& Pius, 2015). Therefore, in vitro assays are preferred as they are rapid, simple and
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particularly useful in determining the effects of processing on bioavailability of
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compounds (Cilla, Bosch, Barberá, & Alegría, 2017). Therefore, in vitro dialyzability
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was used as an index of mineral bioavailability (Luten et al., 1996). Equilibrium dialysis
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method employed in the current study estimates bioavailability by measuring the fraction
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of nutrients that dialyzes through a membrane under simulated gastro-intestinal condition
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(Hemalatha, Platel, & Srinivasan, 2007). In vitro dialyzability is more reliable in
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estimating bioavailability than molar ratios (Liang et al., 2008). In vitro dialyzable Zn in
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the non-fermented and 24 h fermented SCM are shown in Fig. 4a. A significant increase
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in in vitro dialyzable Zn was observed after 24 h fermentation. In vitro dialyzable Zn
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was the highest in fermented NSCM (0.49 mg/100 g). Hydrothermally treated SCM
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which showed least reduction in phytate after fermentation also showed a very little
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increase in bioavailable Zn. We are unaware of any study which measured in vitro
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dialyzable minerals from SCM samples after fermentation with phytase-active
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lactobacilli. However, an increase HCl-soluble Zn was observed in sourdoughs fermented
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with LAB (Anastasio et al., 2010).
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Among fermented samples, Zn bioaccessibility was lowest from fermented
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HTSCM (12.10%), while maximum values were recorded for fermented MSCM
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(34.57%) (Fig.4b). Although bioavailable Zn in fermented NSCM was the highest,
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bioacessibility was maximum in fermented MSCM. This is attributed to the differences in
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the total Zn in NSCM (2.257 mg/100 mg) and MSCM (1.44 mg/100 mg). Therefore, L.
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pentosus CFR3 improved Zn bioacessibility from NSCM and MSCM to greater extent
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than HTSCM.
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Fermentation of autoclaved finger millet SCM with L. pentosus CFR3 resulted in
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significant phyate reduction and improved Zn bioacessibility. The extent and rate of
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phytate hydrolysis is dependent on the nature of the substrate. Therefore, the capacity of
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the test strain to dephytinize individual substrates must be examined. In view of the
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increased Zn bioaccesibility, further improvement has to be achieved through
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optimization of fermentation parameters. However, the study has proved the feasibility
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of using phytase-active L. pentosus CFR3 to dephytinize phytate-rich food ingredients for
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subsequent application in food preparation.
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Conflict of Interest: Authors declare no conflict of interest
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Acknowlegdements: Authors thank the Director, CSIR-CFTRI for providing necessary
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research facilities. AGK acknowledges Council of Scientific and Industrial Research
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(CSIR), New Delhi for the fellowship grant.
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Luten, J., Crews, H., Flynn, A., Van Dael, P., Kastenmayer, P., Hurrell, R. et al (1996).
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Interlaboratory trial on the determination of the in vitro iron dialysability from
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food. Journal of the Science of Food and Agriculture, 72(4), 415–424.
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Ma, G., Jin, Y., Piao, J., Kok, F., Guusje, B., & Jacobsen, E. (2005). Phytate, calcium,
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iron, and zinc contents and their molar ratios in foods commonly consumed in
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China. Journal of Agricultural and Food Chemistry, 53(26), 10285–10290.
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Malleshi, N. G., & Desikachar, H. S. R. (1979). Malting quality of new varieties of ragi
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(Eleusine coracana). Journal of Food Science and Technology, India, 16(4), 149–
366
150.
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Marklinder, I. M., Larsson, M., Fredlund, K., & Sandberg, A.-S. (1995). Degradation of
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phytate by using varied sources of phytases in an oat-based nutrient solution
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fermented by Lactobacillus plantarum strain 299 V. Food Microbiology, 12, 487–
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495.
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Najafi, M. A., Rezaei, K., Safari, M., & Razavi, S. H. (2012). Use of sourdough to reduce
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phytic acid and improve zinc bioavailability of a traditional flat bread (sangak)
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from Iran. Food Science and Biotechnology, 21(1), 51–57.
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Palacios, M. C., Haros, M., Rosell, C. M., & Sanz, Y. (2005). Characterization of an acid
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phosphatase from Lactobacillus pentosus: regulation and biochemical properties.
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Journal of Applied Microbiology, 98(1), 229–237.
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Poutanen, K., Flander, L., & Katina, K. (2009). Sourdough and cereal fermentation in a nutritional perspective. Food Microbiology, 26(7), 693–699. Prasad, A. S. (2012). Discovery of human zinc deficiency: 50 years later. Journal of
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Trace Elements in Medicine and Biology, 26(2), 66–69. Raghavendra, P., & Halami, P.M. (2009). Screening, selection and characterization of
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phytic acid degrading lactic acid bacteria from chicken intestine. International
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Journal of Food Microbiology, 133(1).129-134.
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Raghavendra, P., Ushakumari, S., & Halami, P. (2011). Phytate-degrading Pediococcus
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pentosaceus CFR R123 for application in functional foods. Beneficial Microbes,
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2(1), 57–61.
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Reale, A., Konietzny, U., Coppola, R., Sorrentino, E., & Greiner, R. (2007). The
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importance of lactic acid bacteria for phytate degradation during cereal dough
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fermentation. Journal of Agricultural and Food Chemistry, 55(8), 2993–2997.
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Sabu, A., Augur, C., Swati, C., & Pandey, A. (2006). Tannase production by
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Lactobacillus sp. ASR-S1 under solid-state fermentation. Process Biochemistry,
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41(3), 575–580. Sandberg, A. S., Hulthen, L. R., & Turk, M. (1996). Dietary Aspergillus niger phytase
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increases iron absorption in humans. The Journal of Nutrition, 126(2), 476.
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Zamudio, M., González, A., & Medina, J. A. (2001). Lactobacillus plantarum phytase
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activity is due to non-specific acid phosphatase. Letters in Applied Microbiology,
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32(3), 181–184.
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Legends to Figures & tables
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Fig.1. Dephytinization of SCM with CP from L. pentosus CFR3
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Fig.2. Effect of inoculum size on phytate degradation
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Fig.3. Growth (a), pH reduction (b) and phytate reduction (c) in SCM during 24 h
404
fermentation of SCM with L. pentosus CFR3
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Fig.4. Bioavailability (a) and Bioaccessibility Zn (b) from SCM after 24 h fermentation
406
with L. pentosus CFR3
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Table 1 Phytate, total Zn, total Ca and mineral molar ratios of SCM before and after 24 h
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fermentation with L. pentosus CFR3
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Supplementary file legend
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Fig. S1. Seed coat matter samples used in the study
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Phytate (mg/100g)
Zinc (mg/100g)
Calcium (mg/100g)
PA/Zn
PA/Ca
NSCM
580.03 ± 15.0a
2.257 ± 0.0091a
665.8 ± 10.3a
25.80
0.050
MSCM
384.40 ± 19.0b
1.44 ± 0.0433b
601.15 ± 7.64b
26.40
0.038
HTSCM
730.24 ± 3.99c
2.229 ± 0.0189c
1380 ± 15.23c
36.60
0.031
NSCM#
311.42 ± 0.19d
1.743 ± 0.0176b
810.1 ± 3.76d
18.07
0.023
MSCM#
265.2 ± 1.46d
1.386 ± 0.0319a
749 ± 6.91e
19.20
0.021
HTSCM#
638.97 ± 0.570e
2.818 ± 0.1204d
1600 ± 22.05f
22.32
0.024
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# Fermented Values (mg/100g) are mean ± SD from triplicate analysis (n=3) Different superscript letters in the same column indicate significant difference with Tukey’s test (p<0.05)
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Fig. 1.
buffer (■); wheat phytase (■); crude phytase (□) from L. pentosus CFR3 Results are mean ± SD from triplicate analysis (n=3)
In a group, an asterisk indicates that the mean is statistically different from the negative control (Buffer) by Dunnett's test (p ≤ 0.05).
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Fig. 2.
SCM-based media was inoculated with cell suspension of 9.6X107 CFU/mL and incubated for 16-18 h. 0% (■);1% (■); 3% (■); 5% (■);7% (□) Results are mean±SD from triplicate analysis (n=3). In a group, an asterisk indicates that the mean is statistically different from the uninoculated control (0%) by Dunnett's test (p ≤ 0.05). Different small letters indicate significant difference between the samples in the same group by Tukey's test (p ≤ 0.05).
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NSCM (line); MSCM (dashed lime); HTSCM (dotted line) Results are mean ± SD from triplicate analysis (n=3)
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non-fermented (■); fermented (□)
Results are mean ± SD from triplicate analysis (n=3) Different small letters indicate significant difference between the samples in the same group by Tukey's test (p ≤ 0.05)
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Finger millet seed coat matter (SCM) is a byproduct of millet milling.
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SCM is rich in minerals but also contain phytate.
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SCM was fermented with phytase-active Lactobacillus pentosus CFR3.
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Reduction in phytate and increase in zinc bioaccessibility was noted.
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Dephytinized SCM may be used as food ingredients.
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S0023-6438(17)30699-0
DOI:
10.1016/j.lwt.2017.09.024
Reference:
YFSTL 6539
To appear in:
LWT - Food Science and Technology
Received Date: 25 May 2017 Revised Date:
24 August 2017
Accepted Date: 15 September 2017
Please cite this article as: Amritha, G.K., Dharmaraj, U., Halami, P.M., Venkateswaran, G., Dephytinization of seed coat matter of finger millet (Eleusine coracana) by Lactobacillus pentosus CFR3 to improve zinc bioavailability, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.09.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dephytinization of seed coat matter of Finger Millet (Eleusine coracana) by
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Lactobacillus pentosus CFR3 to improve Zinc bioavailability
3 Authors: Amritha Girish K1, Usha Dharmaraj2, Prakash M. Halami1, and G.
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Venkateswaran1*
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Microbiology and Fermentation Technology Department
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Grain Science Technology Department
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CSIR-Central Food Technological Research Institute
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Mysuru-570 020
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Karnataka, India
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G. Venkateswaran
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Chief Scientist & Head
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Microbiology and Fermentation Technology
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CSIR- Central Food Technological Research Institute
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Mysuru-570 020
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Karnataka, India
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Email: [email protected] /[email protected]
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Abstract
25 Finger millet seed coat matter (SCM) is a rich source of dietary fiber, calcium, iron and
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zinc (Zn). However, the bioavailability of minerals especially Zn is limited as it occurs as
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insoluble complexes with phytate. This study envisages the potential of phytase-active
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Lactobacillus pentosus CFR3 to dephytinize SCM derived from native (non-processed),
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malted and hydrothermally treated finger millets with an expectation to improve its Zn
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bioavailability. After 24 h fermentation, the phytate in native, malted and hydrothermally
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treated SCM reduced to 56.70%, 66.65% and 87.85% respectively. Correspondingly,
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Phytate/Zn molar ratios decreased to 18.20, 19.20 and 22.32 indicating improved Zn
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bioavailability. Also, Zn bioacessiblity from native, malted and hydrothermally treated
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SCM increased to 28.40%, 34.57% and 12.10 % as determined by in vitro dialyzability
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experiments following 24 h fermentation. Therefore, fermentation of SCM with L.
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pentosus CFR3 represents a safe and feasible solution to counteract the effects of phytate
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on Zn absorption.
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Keywords: Seed coat; finger millet; Lactobacillus pentosus; dephytinize; Zinc
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1. Introduction
48 The seed coat matter (SCM) from native, malted and hydrothermally treated finger millet
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is a byproduct of the millet processing industry. It contains significant proportion of
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minerals, dietary fiber and phytochemicals. In the recent years, SCM has been utilized as
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adjuncts in cereal foods to obtain products with high levels of dietary fiber, calcium (Ca),
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iron (Fe) and zinc (Zn) (Krishnan, Dharmaraj, & Malleshi, 2012; Krishnan, Dharmaraj,
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Manohar, & Malleshi, 2011). However, the bioavailability of minerals from SCM may be
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limited due to the presence of phytate (myo-inositol hexakisphosphate) (Poutanen,
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Flander, & Katina, 2009). The phosphate groups of phytate form stable complexes with
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dietary cations, thus hindering their bioavailability (Liang, Han, Nout, & Hamer, 2008).
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Zinc bioavailability is most severely affected by dietary phytate (Lönnerdal, 2002;
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Lopez, Leenhardt, Coudray, & Remesy, 2002). Several studies have revealed a positive
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correlation between phytate-rich diets and Zn deficiency in humans (Lönnerdal, 2002).
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Clinical manifestations of Zn deficiency include growth retardation, hypogonadism in
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males, rough skin, impaired immunity, neuro-sensory disorder and cognitive impairment
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(Lönnerdal, 2002; Prasad, 2012). Therefore, optimization of Zn bioavailability is of
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significance.
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Enzymatic phytate hydrolysis is a promising strategy to improve Zn absorption in
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humans (Fischer, Egli, Aeberli, Hurrell, & Meile, 2014). Food ingredients have been
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successfully dephytinized with phytate-degrading enzymes (phytases) (Haros, Rosell, &
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Benedito, 2001; Marklinder, Larsson, Fredlund, & Sandberg, 1995; Sandberg, Hulthen,
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& Turk, 1996). Several researchers have documented the presence of phytases in Lactic
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Acid Bacteria (LAB) (De Angelis et al., 2003; Palacios, Haros, Rosell, & Sanz, 2005;
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Raghavendra & Halami, 2009; Zamudio, González, & Medina, 2001). This opens up
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possibilities towards fermentative reduction of phytate with specific phytase-active LAB.
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The application of LAB to dephytinize food ingredients is safe due to the GRAS
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(Generally Regarded as Safe) status conferred on this bacterial group. Moreover, it is
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cheaper compared to the use of purified phytase.
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Elimination of phytate from byproducts of millet milling with specific LAB is a
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relatively unexplored area of research. Therefore, the current investigation focused on the
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dephytinization of SCM from native, malted and hydrothermally treated finger millets by
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Lactobacillus pentosus CFR3 as a means to improve Zn bioacessibility.
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2. Materials and methods
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2.1. Sample preparation
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Finger millet (GPU 28 Variety) was procured from University of Agriculture Sciences,
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Bangalore, Karnataka, India. Native SCM (NSCM) was prepared as described by
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Chethan and Malleshi (2007). Malted SCM (MSCM) was obtained following the protocol
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given by Malleshi and Desikachar (1979), while hydrothermally treated SCM (HTSCM)
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was prepared as described by Krishnan et al. (2012). The SCM samples were finely
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powdered in a laboratory grinder, passed through a 60 BSS mesh and stored at 4 ºC until
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analysis.
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2.2. Materials
94 Dialysis membrane (10 kDa cut-off), hydrogen peroxide (H2O2; 30%), lanthanum
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chloride (LaCl3), lysozyme, mineral standards (Ca and Zn), nitric acid (HNO3; 90%),
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sodium phytate and wheat phytase were from Sigma (St Louis, MO, USA).
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Microbiological media components, pancreatin, pepsin and ox-bile were purchased from
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Hi Media (Mumbai, India). All other reagents of analytical grade were obtained from
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Sisco Research Laboratories (India). Deionized water from Millipore system and acid-
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washed glassware were used for the entire study.
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2.3. Bacterial strain
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Lactobacillus pentosus CFR3 which produced phytate-degrading enzyme was used in the
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study (Amritha, Halami, & Venkateswaran, 2017). The culture was maintained at -20 ºC
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in MRS (De Man, Ragosa and Sharpe) broth with 40% (v/v) glycerol. The strain was
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activated from the glycerol stock by subculturing in MRS broth twice.
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Lactobacillus pentosus CFR3 was propagated in modified MRS broth (pH 6.5 ± 0.2)
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under static condition for 8 h at 37 ºC. Modified MRS broth was prepared by replacing
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glucose with maltose. Also, sodium di-hydrogen phosphate (KH2PO4) was replaced with
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sodium phytate (0.65 g/L) and 0.1 M 3-[N-morpholino] propanesulfonic acid (MOPS).
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The cell-associated phytase from the culture was extracted with lysozyme and NaCl
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(Amritha et al., 2017).
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2.5 Dephytinization of SCM with CP of L. pentosus CFR3
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The SCM samples (0.5 g) were suspended in 2 mL of CP (~8 mg protein) and incubated
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in a shaking water bath at 50 ºC for 120 min. Similarly, samples suspended in 0.25%
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(w/v) wheat phytase (0.01 - 0.04 U/mg) in sodium acetate buffer (0.1 M, pH 5.5) and
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sodium acetate buffer constituted the positive and negative controls respectively. Post
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incubation; samples were freeze-dried (Freeze Zone Freeze dry System, Labconco, USA)
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and subjected to phytate extraction and estimation.
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2.6. Inoculum preparation and fermentation of SCM with L. pentosus CFR3
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Lactic fermentation of SCM were carried out based on the procedure given by Reale,
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Konietzny, Coppola, Sorrentino, and Greiner (2007). Lactobacillus pentosus CFR3 was
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propagated in MRS for 16 ± 2 h at 37 ºC. Cells were harvested (7000 g, 10 min, 4 ºC),
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washed and resuspended in an equal volume of saline to obtain a cell suspension (108
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CFU/mL). The SCM samples were autoclaved (15 psi, 121 ºC, 30 min) and subsequently
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suspended in sterile water under aseptic conditions to obtain a 10% (w/v) suspension. The
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SCM-based suspensions were inoculated with saline suspension of the producer strain
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and incubated at 37 ºC in a shaker incubator (New Brunswick, USA) at 150 rpm.
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Uninoculated control was run in parallel.
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2.7. Determination of viable cell counts and pH
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incubated at 37 ºC for 24-48 h. Changes in pH of the fermenting SCM was noted with a
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pH meter (Eutech Instruments, Singapore).
144 2.8. Chemical assays
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Phytate was extracted and estimated from SCM samples as described by Gao et al. (2007)
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with sodium phytate as the standard.
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Total minerals (Ca and Zn) were estimated according to Kumari and Platel (2016) with
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minor modifications. Briefly, samples (0.1 g) were wet digested for 24 h with 0.5 mL
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HNO3 and 0.3 mL H2O2, followed by gradual refluxing in a hot plate. Subsequently, the
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volume was made up to 10 mL with 0.5% HNO3. The samples were filtered through 0.45
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µm syringe filters (Millex HV, Millipore) and appropriately diluted before estimation
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using Flame Atomic Absorption Spectrometry (FAAS; Shimadzu AAF-6701). For Ca
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estimation, the samples and standards were spiked with 0.1% (w/v) LaCl3 to prevent
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phosphorous interference.
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2.8.2. In vitro dialyzable Zn
163 In vitro dialyzable Zn was determined by equilibrium dialysis method as described by
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Luten et al. (1996) with modifications. In short, samples (10 g) were suspended in 80 mL
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water and pH was adjusted to 2.0 with 6.0 M HCl. Subsequently, pepsin solution (3 mL
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of 16 g/100 mL pepsin in 0.1 M HCl) was added to the mixture and the volume was
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raised to 100 mL. The mixture was incubated in a shaker at 37 ºC for 2 h at 110 rpm to
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obtain gastric digests. The gastric digests were frozen for 30 min to stop the reaction.
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Titratable acidity (TTA) was measured in an aliquot (20 mL) of the gastric digest
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containing 5 ml of pancreatin-bile mix (5 g pancreatin + 25 g ox-bile in 1 L of 0.1 M
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sodium bicarbonate (NaHCO3)) by adjusting the pH to 7.5 with 0.2 M sodium hydroxide
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(NaOH). An aliquot of gastric digest (20 mL) was now subjected to intestinal digestion in
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a beaker containing dialysis membrane filled with 25 mL of NaHCO3 equal in moles to
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the NaOH as determined by TTA. The beakers with the dialysis membranes were
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incubated in a shaker as described above until the pH of the digest changed to 5.0.
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Pancreatin-bile mix was added (5 mL) and the incubation was continued until the pH
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reached 7.0. After incubation, dialysis membranes were rinsed with distilled water and its
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volume was noted. Further, the dialysates (bioavailable fraction) were acidified with
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HNO3 at 5% level, centrifuged (10,000 g) and filtered before estimating the Zn content
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by AAS. Bioacessibility was calculated as follows
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Bioaccessibility (%) = 100 x Y/Z where,
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Y is the Zn content in bioavailable fraction (mg of mineral element/ 100 g sample) and Z
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is the total Zn (mg mineral element/100 g sample).
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2.9. Statistical analysis
186 Results were expressed as mean of triplicates ± standard deviation (SD). Testing for
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statistical significance was done by one-way ANOVA. Post hoc comparison was
189
performed by Dunnet's and Tukey's tests. Differences were considered significant
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when p <0.05. All analyses were performed using GraphPad Prism 7 (GraphPad Software
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Inc., San Diego, CA, USA).
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3. Results and Discussion
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3.1. Dephytinization of SCM by treatment with CP
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Seed coat matter from native, malted and hydrothermally treated finger millets have been
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used as adjuncts in the preparation of cereal-based food (Krishnan et al., 2011).
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Therefore, we examined the ability of L. pentosus CFR3 to dephytinize all three types of
200
SCM. To emphasize enzymatic nature of phytate reduction, NSCM, MSCM and HTSCM
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were treated with CP from the test strain. Under given conditions, phytate in NSCM,
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MSCM and HTSCM reduced to 86.75%, 89.39% and 96.03% respectively (Fig. 1). The
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observed reduction in phytate was attributed to CP as the substrates were autoclaved to
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rule out plant phytases if any. A more pronounced phytate reduction was noted when
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CP from L. pentosus CFR3 was used to dephytinize whole finger millet flour (Amritha et
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al., 2017).
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3.2. Fermentative reduction of phytate from SCM
209 Removal of phytate through fermentation is simpler and cost-effective compared to the
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use of purified phytases. Therefore, further studies were in this direction. Phytate-
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degrading LAB has been successfully grown in cereal-based substrates with concomitant
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phytate reduction (Anastasio et al., 2010; Raghavendra, Ushakumari, & Halami, 2011).
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However, there are no reports on fermentative reduction of phytate from finger millet
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SCM by L. pentosus. Further, the major nutrients in all three SCM samples were
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comparable (Krishnan et al., 2011). The initial pH of NSCM and HTSCM was similar
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(5.60-5.8) while that of MSCM was slightly lower (~4.80).
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Inoculum size standardization was carried out by fermenting SCM with varying
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levels of inoculum for 16 ± 2 h (Fig. 2). A positive correlation was observed between
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inoculum size and phytate reduction. Since reduction rates obtained with 5% and 7%
221
inoculum were statistically insignificant (p <0.05), 5% inoculum was used for time
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course experiments. Lower inoculum levels may not be adequate for initiating growth
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and enzyme synthesis on different substrates. In general, increased inoculum size
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enhances microbial growth and enzyme synthesis to a certain level. A balance between
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the proliferating bacterial biomass and the fermentation substrates is critical for optimal
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enzyme activity (Sabu, Augur, Swati, & Pandey, 2006).
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Growth of L. pentosus CFR3 and evolution of pH during 24 h fermentation of
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SCM is shown in Figs. 3a and 3b. Initial phytate content of NSCM, MSCM and HTSCM
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were 580.03, 384.40 and 730.24 mg/100 g respectively. After 24 h fermentation, phytate
230
levels dropped to 56.7%, 66.65% and 87.85% (Fig. 3c). Phytate reduction in NSCM was
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gradual, while phytate in MSCM decreased drastically within 3 h after which significant
232
hydrolysis was not observed. In case of HTSCM, phytate reduction was less compared to
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NSCM and MSCM. The unique patterns of phytate degradation observed with the three
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SCM samples may be due to the differences in initial phytate concentration and the
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nature of phytate (Lan, Abdullah, Jalaludin, & Ho, 2002). Interestingly, HTSCM which
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did not show much phytate degradation showed a similar trend when treated with CP.
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3.3. Molar ratios of phytic acid to zinc (PA/Zn)
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Phytate hydrolysis is likely to improve Zn bioavailability as it is the single major
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inhibitor of Zn bioavailability (Lönnerdal, 2002). Phytate to zinc molar ratio is assumed
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to provide an indication of the Zn bioavailability, with PA/Zn molar ratios>15 hinting
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towards reduced Zn absorption (Liang et al., 2008; Ma et al., 2005). Although a decrease
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in PA/Zn values were observed for 24 h fermented SCM (Table 1), they were slightly
245
above the critical threshold. However, an increase in Zn bioavailability is expected.
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Several researchers have reported reduced PA/Zn molar ratios in cereal and legume
247
based food substrates after lactic fermentation (Fischer, Egli, Aeberli, Hurrell, & Meile,
248
2014; García-Mantrana, Monedero, & Haros, 2015; Najafi, Rezaei, Safari, & Razavi,
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2012). Since finger millet SCM is very rich in Ca (Krishnan et al., 2012), PA/Ca ratios
250
were also calculated. Calcium bioavailability is compromised when PA/Ca exceeds 0.24
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(García-Mantrana et al., 2015; Ma et al., 2005). In this context, calcium bioavailability
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from none of the samples is likely to be impaired.
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3.4. In vitro dialyzability of Zinc
255 Ideally bioavailability studies should be carried out in vivo in human subjects because
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data from animal models are questionable due to differences in the physiology between
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humans and model organism. However, human trials are expensive and difficult (Amalraj
259
& Pius, 2015). Therefore, in vitro assays are preferred as they are rapid, simple and
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particularly useful in determining the effects of processing on bioavailability of
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compounds (Cilla, Bosch, Barberá, & Alegría, 2017). Therefore, in vitro dialyzability
262
was used as an index of mineral bioavailability (Luten et al., 1996). Equilibrium dialysis
263
method employed in the current study estimates bioavailability by measuring the fraction
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of nutrients that dialyzes through a membrane under simulated gastro-intestinal condition
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(Hemalatha, Platel, & Srinivasan, 2007). In vitro dialyzability is more reliable in
266
estimating bioavailability than molar ratios (Liang et al., 2008). In vitro dialyzable Zn in
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the non-fermented and 24 h fermented SCM are shown in Fig. 4a. A significant increase
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in in vitro dialyzable Zn was observed after 24 h fermentation. In vitro dialyzable Zn
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was the highest in fermented NSCM (0.49 mg/100 g). Hydrothermally treated SCM
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which showed least reduction in phytate after fermentation also showed a very little
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increase in bioavailable Zn. We are unaware of any study which measured in vitro
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dialyzable minerals from SCM samples after fermentation with phytase-active
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lactobacilli. However, an increase HCl-soluble Zn was observed in sourdoughs fermented
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with LAB (Anastasio et al., 2010).
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Among fermented samples, Zn bioaccessibility was lowest from fermented
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HTSCM (12.10%), while maximum values were recorded for fermented MSCM
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(34.57%) (Fig.4b). Although bioavailable Zn in fermented NSCM was the highest,
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bioacessibility was maximum in fermented MSCM. This is attributed to the differences in
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the total Zn in NSCM (2.257 mg/100 mg) and MSCM (1.44 mg/100 mg). Therefore, L.
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pentosus CFR3 improved Zn bioacessibility from NSCM and MSCM to greater extent
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than HTSCM.
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282 4. Conclusion
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Fermentation of autoclaved finger millet SCM with L. pentosus CFR3 resulted in
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significant phyate reduction and improved Zn bioacessibility. The extent and rate of
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phytate hydrolysis is dependent on the nature of the substrate. Therefore, the capacity of
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the test strain to dephytinize individual substrates must be examined. In view of the
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increased Zn bioaccesibility, further improvement has to be achieved through
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optimization of fermentation parameters. However, the study has proved the feasibility
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of using phytase-active L. pentosus CFR3 to dephytinize phytate-rich food ingredients for
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subsequent application in food preparation.
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Conflict of Interest: Authors declare no conflict of interest
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Acknowlegdements: Authors thank the Director, CSIR-CFTRI for providing necessary
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research facilities. AGK acknowledges Council of Scientific and Industrial Research
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(CSIR), New Delhi for the fellowship grant.
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Legends to Figures & tables
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Fig.1. Dephytinization of SCM with CP from L. pentosus CFR3
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Fig.2. Effect of inoculum size on phytate degradation
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Fig.3. Growth (a), pH reduction (b) and phytate reduction (c) in SCM during 24 h
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fermentation of SCM with L. pentosus CFR3
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Fig.4. Bioavailability (a) and Bioaccessibility Zn (b) from SCM after 24 h fermentation
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with L. pentosus CFR3
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Table 1 Phytate, total Zn, total Ca and mineral molar ratios of SCM before and after 24 h
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fermentation with L. pentosus CFR3
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Supplementary file legend
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Fig. S1. Seed coat matter samples used in the study
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Phytate (mg/100g)
Zinc (mg/100g)
Calcium (mg/100g)
PA/Zn
PA/Ca
NSCM
580.03 ± 15.0a
2.257 ± 0.0091a
665.8 ± 10.3a
25.80
0.050
MSCM
384.40 ± 19.0b
1.44 ± 0.0433b
601.15 ± 7.64b
26.40
0.038
HTSCM
730.24 ± 3.99c
2.229 ± 0.0189c
1380 ± 15.23c
36.60
0.031
NSCM#
311.42 ± 0.19d
1.743 ± 0.0176b
810.1 ± 3.76d
18.07
0.023
MSCM#
265.2 ± 1.46d
1.386 ± 0.0319a
749 ± 6.91e
19.20
0.021
HTSCM#
638.97 ± 0.570e
2.818 ± 0.1204d
1600 ± 22.05f
22.32
0.024
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Sample
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Table 1
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# Fermented Values (mg/100g) are mean ± SD from triplicate analysis (n=3) Different superscript letters in the same column indicate significant difference with Tukey’s test (p<0.05)
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Fig. 1.
buffer (■); wheat phytase (■); crude phytase (□) from L. pentosus CFR3 Results are mean ± SD from triplicate analysis (n=3)
In a group, an asterisk indicates that the mean is statistically different from the negative control (Buffer) by Dunnett's test (p ≤ 0.05).
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Fig. 2.
SCM-based media was inoculated with cell suspension of 9.6X107 CFU/mL and incubated for 16-18 h. 0% (■);1% (■); 3% (■); 5% (■);7% (□) Results are mean±SD from triplicate analysis (n=3). In a group, an asterisk indicates that the mean is statistically different from the uninoculated control (0%) by Dunnett's test (p ≤ 0.05). Different small letters indicate significant difference between the samples in the same group by Tukey's test (p ≤ 0.05).
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Fig. 3.
NSCM (line); MSCM (dashed lime); HTSCM (dotted line) Results are mean ± SD from triplicate analysis (n=3)
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Fig.4.
non-fermented (■); fermented (□)
Results are mean ± SD from triplicate analysis (n=3) Different small letters indicate significant difference between the samples in the same group by Tukey's test (p ≤ 0.05)
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Finger millet seed coat matter (SCM) is a byproduct of millet milling.
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SCM is rich in minerals but also contain phytate.
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SCM was fermented with phytase-active Lactobacillus pentosus CFR3.
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Reduction in phytate and increase in zinc bioaccessibility was noted.
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Dephytinized SCM may be used as food ingredients.
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•