This document discusses increasing micronutrient bioavailability in foods through the application of phytases. It notes that micronutrient deficiencies are common in developing countries where diets rely heavily on cereals, which contain antinutritional factors that reduce bioavailability. Phytase enzymes can help address this issue by breaking down phytic acid, one of the main inhibitors. The document then provides details on the isolation and characterization of fungal phytases, and their effects on increasing the dialyzability of minerals like iron and zinc from biofortified wheat. It concludes by discussing opportunities to further improve micronutrient bioavailability through probiotic microbes or consumer-acceptable applications of phytases.

1. Increasing micronutrient
bioavailability in foods by phytase
applications
Vinod Kumar, Ph.D.
Assistant Professor
Department of Biotechnology
Akal College of Agriculture, Eternal University, Baru Sahib (H.P.)
Email: sangwan.vinod@yahoo.com;
Webpage: https://www.researchgate.net/profile/Vinod_Kumar133/
Google Scoholar link: http://scholar.google.com/citations?hl=en&user=dQVRZlEAAAAJ
National Conference, Shoolini University,
Solan, 02-03 March, 2017

2. Micronutrient Bioavailability: Issue and
challenges
• Hidden hunger in 2/3rd world population
• Dependence on cereals as foods
• Biofortification can increase micronutrient
concentration but the bioavailability is still of
great concern
• Low due to higher antinutritional factors in
cereals
• Fe and Zn deficiencies are most concerned
• Food/feed enzyme may be used during
processing
National Conference, Shoolini University, Solan, March 3, 2017

3. Factors affecting micronutrient
bioavailability
• Inhibitors
Phytic acid
Tannic acid
Dietary fibers
Calcium
• Enhancers
Ascorbic acid
Inorganic phosphate
β-carotene
National Conference, Shoolini University, Solan, March 3, 2017

4. Phytic Acid
• Phosphorus is found in plant seeds as primary storage form,
called as phytic acid or phytate (Brinch-Pederson at al., 2012)
• It consist of an inositol (chair configuration) ring, known as
IP6, myo- inositol-1, 2, 3, 4, 5, 6 – hexakisphophate
• In plants, it is found as anionic in nature
• IP6 form complexes with Ca, Mg and K, in some cases it also
bound to starches and proteins (Vat and Banerjee., 2004)
• This chelated molecule of IP6, commonly known as phytin
(Lott et al., 1984)
• In grains, 90% of the seeds phytic acid is found in the aleurone
layer and remaining 10% in the scutellum.
National Conference, Shoolini University, Solan, March 3, 2017

5. Phytase: What does it do???
National Conference, Shoolini University, Solan, March 3, 2017
Phytase classes:
1. Beta propeller phytase (BPPhy)
2. Histidine acid phytase (HAPhy)
3. Purple acid phytase (PAPhy)
4. Cysteine phytase (CyPhy)

6. Phytase application: current approaches???
Activation of endogenous phytase by germination
Exogenous addition of microbial phytase
Developing transgenic plants with phytase gene to
produce low phytate seeds
Direct co-cultivation/application of phytase producing
probiotic microbes
National Conference, Shoolini University, Solan, March 3, 2017

7. DBT Network project
Screening iron, zinc and carotenoid
bioavailability from biofortified staple crops
using coupled in vitro digestion/Caco-2 cell
model
• Eternal University, Baru Sahib
• Analysis of biofortified cereals for various inhibitors and their
correlation with dialyziability values of zinc and iron.
• Characterization of fungal phytases and effect of its supplementation on
zinc and iron dialyzability using in vitro dialyzability and caco2 cell line
model.
• National Institute of Nutrition, Hyderabad
• Indian Institute of Technology, Roorkee
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8. S. No. Fungi Isolation Source Identified as Phytase activity (U/g)
1 EUFR-1 Wheat Penicillium sp. 7.988
2 EUFR-2 Chilli seeds Talaromyces sp. 9.140
3 EUFR-3 Soil Penicillium oxalicum 12.80
4 EUFR-4 Poultry soil Trichoderma sp. 0.707
5 EUFR-5 Guggal Aspergillus ochraceus 1.930
6 EUFR-6 Maize Rhizosphere Aspergillus sp. 0.085
7 EUFR-7 Bajra Rhizosphere Aspergillus sp. 4.903
8 EUFR-8 Shilazit Trichoderma sp. 0.058
9 EUFR-9 Shilazit Actinomycetes sp. 0.150
10 EUFR-10 Bajra seed Tolyposporium sp. 0.781
11 EUFR-11 Soybean Rhizosphere Actinomycetes sp. 1.098
12 EUFR-12 Infected seed of flower Alternaria sp. 0.903
13 EUFR-13 Parthenium plant Alternaria sp. 0.058
14 EUFR-14 Plant bark Yeast sp. 0.142
15 EUFR-15 Parthenium plant Penicillium crustosum 1.220
16 EUFR-16 Infected wheat plant Ustilago sp. 10.09
17 EUFR-17 Soil Penicillium sp. 4.718
18 EUFR-18 Soil Aspergillus sp. 2.951
19 EUFR-19 Bajra Rhizosphere Aspergillus sp. 5.951
20 EUFR-20 Soybean Rhizosphere Aspergillus sp. 1.171
21 EUFR-21 Wax Penicillium sp. 2.075
22 EUFR-22 Wax Actinomycetes sp. 0.488
23 EUFR-23 Plant bark Penicillium sp. 0.258
24 EUFR-24 Wood Trichoderma viridae 7.157
25 EUFR-25 Wood Cephalosporium sp. 0.403
26 EUFR-26 Asrol Penicillium sp. 0.703
27 EUFR-27 Lab contaminated media Aspergillus sp. 0.988
28 EUFR-28 Garlic covering Alternaria sp. 1.313
29 EUFR-29 Badam Cladosporium sp. 0.039
30 EUFR-30 Lab contaminated media Cladosporium sp. 0.077
31 EUFR-31 Wood Trichoderma sp. 1.659
32 EUFR-32 Plant leaf Penicillium sp. 1.198
33 EUFR-33 Kari patta leaves Secletoria sp. 1.269
34 EUFR-34 Wood Stachybotrys sp. 1.622
35 EUFR-35 Wood Penicillium sp. 0.058
36 EUFR-36 Wood Cephalosporium sp. 2.727
37 EUFR-37 Shilazit sample Stachybotrys sp. 0.806
38 EUFR-38 Maize Rhizosphere Aspergillus sp. 0.064
39 EUFR-39 Baker yeast Yeast sp. 0.180
40 EUFR-40 Zymograma bicalrata Beetle Penicillium sp. 0.012
Isolation sources, morphological identification and phytase activity of 40 microbial isolates
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9. National Conference, Shoolini University, Solan, March 3, 2017

10. Samples Dialyzable micronutrients (ppm)
Cu Mn Fe Zn Ca
PRH3-30-3 2.75 28.6 4 16.71 1667.5
PRH3-43-1 2.61 34.75 4.35 21.425 2897.5
PRH3-78-1-3 3.2 31.8 6.6 18.37 2012.5
PRH3-15-12 2.45 22.75 7.9 18.015 1815
46-1-15-15-3-1 2.95 36.35 4 38.06 647.5
MB-16-1-4 3.1 33.2 -1.35 21.88 1630
MB-2-3 3.2 15.25 0.05 15.025 3947.5
PRH2-487-6-7 14.25 34.55 2.75 26.205 1285
49-1-11-9-7-1 2.4 19.8 -1.85 23.465 1572.5
17-1-2-5-4-9-3 2.75 25.55 0.75 20.155 1517.5
17-1-2-5-4-6-5 2.8 28.15 0.7 21.83 1482.5
PBW343+GPC+LR24 3.25 26.95 -1.45 10.105 1625
HD2967+YR40+LR57 1.15 -1.55 -3.25 0.01 1572.5
Effect of Penicillium oxalicum EUFR3 phytase dephytination treatment
On Micronutrient dialyzability from biofortified wheat flour
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11. Conclusion & future perspectives
• Effectiveness of supplemental phytases in
enhancing the micronutrient bioavailability is
well accepted
• Further research is being required for reducing
the variability of responses.
• Probiotic microbes with suitable phytase activity
• Consumer concern for use of microbial phytases
and maintaining traditional food habits must be
addressed
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12. Acknowledgements
DBT, GoI for providing financial support (Grant No.
BT/AGR/BIOINFO/PHII/NIN/2011)
MoFPI, GoI for infrastructure development grant (F. No.
5-11/2010-HRD)
Prof. H.S. Dhaliwal, Vice Chancellor, Eternal University,
Baru Sahib for providing research infrastructure,
opportunities and motivation
My research team: Sheetal Chanyal, Abhishake Saxena,
Shailja Verma, Imran Sheikh, Ramandeep Kaur, Harpreet Kaur

13. Related Publications
1. Vinod Kumar, Gopal Singh, A K Verma, Sanjeev Agrawal: In silico characterization of
histidine Acid phytase sequences. Enzyme research. 2012; 2012:1-8; 845465.
2. Vinod Kumar, Punesh Sangwan, A. K. Verma, Sanjeev Agrawal: Molecular and biochemical
characteristics of recombinant β-propeller phytase from Bacillus licheniformis
strain PB-13 with potential application in aquafeed. Applied Biochemistry and
Biotechnology, 2014, 173(2):646-659
3. Vinod Kumar, Punesh Sangwan, Gopal Singh, A. K. Verma, Sanjeev Agrawal: Cloning,
sequencing, expression and in silico analysis of β-propeller phytase from Bacillus
licheniformis strain PB-13. Biotechnology Research International, 2014, 2014: 1-11, Article
ID 841353, doi:10.1155/2014/841353
4. Vinod Kumar, Sanjeev Agrawal: An insight into protein sequences of PTPlike cysteine
phytases. Nusantara Bioscience, 2014, 6(1):97-101
5. Harpreet Kaur Kalsi, Rajveer Singh, Harcharan Singh Dhaliwal, Vinod Kumar: Phytases from
Enterobacter and Serratia species with desirable characteristics for food and feed
applications. 2016, 3 Biotech, 6:64
6. Ramandeep Kaur, Abhishake Saxena, Punesh Sangwan, Ajar Nath Yadav, Vinod Kumar*,
Harcharan Singh Dhaliwal,: Production and characterization of a neutral phytase of
Penicillium oxalicum EUFR-3 isolated from Himalayan region. 2017, Nusantara
Bioscience, 9(1): 68-76
7. Vinod Kumar*, Ajar Nath Yadav, Priyanka Verma, Punesh Sangwan, Abhishake Saxena, Krishan
Kumar, Bijender Singh: β-Propeller phytases: Diversity, catalytic attributes, current
developments and potential biotechnological applications, International Journal of
Biological Macromolecules, 2017, DOI: 10.1016/j.ijbiomac.2017.01.134

14. Thanks