Utilization of agro-industrial by-products for the production of kojic acid
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Date
2021
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Punjab Agricultural University, Ludhiana
Abstract
The present study involves a novel fermentative bioprocess optimized for the production of kojic
acid (2-hydroxymethyl-5-hydroxy-γ-pyrone) to combat the problem of oxidative stress posed by
its chemically synthesized forms. Four promising kojic acid producing fungal strains (KJPAU1,
KJPAU2, KJPAU3, KJPAU4) were screened with the Rapid Bio Oxidation (RaBiO) method (91.3
% isolation rate) with the yield of 40.13, 21.1, 9.20, 14.6 gL-1 respectively on GYE medium. The
isolates showed tolerance to the wide range of temperature (20-50 °C), pH (3.5-6.5), assimilated
Carbohydrates (61.9 %), utilized Nitrogen sources (66.67 %), induced hydrolysis of starch,
cellulose. Based on 18SrRNA sequencing, atoxicogenic KJPAU1, KJPAU2 isolates were
characterized as Aspergillus nomius (MH752445), A. oryzae (EF568052) respectively with a >98
% similarity values. Among the agro-industrial substrates (Kinnow residue, Amla waste,
Sugarcane bagasse, Wheat straw) studied for kojic acid production after 10 days of fermentation
with the inoculation rate @ 20 % having the final concentration of 3×105 spores/mL with
intermittent shaking conditions, the parameters of optimized fermentative bioprocess consisted of
Kinnow waste residue (20 %), LCUM (4 %), MgSO4 (0.1 %), KCl (0.05 %), pH 6.0, temperature
30 °C with the maximum production of 11.64 gL-1. The kojic acid productivity in Kinnow waste
was found to be higher, 66.7 %, 16.6 % and 52.1 % from Wheat straw, Amla pomace, Sugarcane
bagasse residues respectively. The maximum activity of Glucose dehydrogenase (44.3 µmol TPF
h-1) at optimized production conditions was estimated on tenth day at pH (6.0), temperature (30
°C), KCl (0.05 %). The downstream processing of kojic acid with the direct concentration and
crystallization method showed >42.23 % recovery with the 97 % purity. The TLC solvent system,
Toluene: Ethyl Acetate: Formic Acid= 3:6:1 facilitated the separation at Rf value (0.64), detected
under UV lamp (336 nm). The purity of fermentatively produced kojic acid was confirmed by C18
RP-HPLC with the Acetonitrile: Phosphate buffer (50:50) as the mobile phase at a flow rate 0.3
mL/ min, with the retention time of 9.143 min (approx.) at 254 nm. With the FTIR spectrum for
functional groups at 3270.8 cm-1, 3179.43 cm-1 (- OH), 2925.17 cm-1, 2854.05 cm-1 (aliphatic-
CH), 1660.59 cm-1 (cyclic-C=O), 1611.11 cm-1 (C=C), 1472.61 cm-1 (deformation of-CH2),
1074.04 cm-1 (cyclic C-O-C), 943.58 cm-1 , 863.66 cm-1 and 775.65 cm-1 (1, 4 α -disubstituted
ring) confirmed the crystalline purity (97 %) of kojic acid. To prevent pericarp browning of litchi
fruit, 8mM kojic acid application for five minutes delayed melanosis up to nine days. The results
of oral fed kojic acid dosage @ 1, 2.5, 5 % in albino mice for 28 days showed no adverse effects
on body weight (g), food/ water intake, general behavior.
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Citation
Sharma,Neha (2021). Utilization of agro-industrial by-products for the production of kojic acid (Unpublished Ph.D. Dissertation). Punjab Agricultural University, Ludhiana, Punjab, India.