Kojic Acid Production Research Articles

Production of Kojic Acid by Aerobic Aspergillus Fermentation

Production of Kojic Acid by Aerobic Aspergillus Fermentation

Effects of dual impeller system of Rushton turbine, concave disk turbine and their combinations on the performance of kojic acid fermentation by Aspergillus flavus Link 44‐1

AbstractIn the present work, the effects of dual impeller configurations comprising of Rushton turbine (RT) and Concave‐bladed disk turbine (CD‐6) on the oxygen transfer profile and fermentation performance of kojic acid production by Aspergillus flavus Link 44‐1 was investigated. Batch cultivations of A. flavus Link 44‐1 were performed in 2 L stirred tank bioreactor. The fermentations were conducted using different dual impeller systems; (1) RT‐RT, (2) CD6‐CD6, (3) RT‐CD6, and (4) CD6‐RT (bottom‐top impeller). It was perceived that dual CD‐6 system was able to improve oxygen transfer rate by about 25–45% over the hybrids of RT and CD‐6 and the typically configured dual RT system. While no substantial disparity could be seen on the fungal growth rate by the manipulation of the impeller, high concentration of kojic acid (44.93 g L−1) was attained with the use of dual CD‐6 as the mixer. Efficient agitating system that can facilitate good gas dispersion capability is crucially required in order to counteract the problem of oxygen solubility limitation faced in such viscous fungal fermentation broth. The results from this work suggested the promising capability of dual CD‐6 configuration in enhancing productivity of kojic acid fermentation in stirred tank bioreactor. © 2014 Curtin University of Technology and John Wiley & Sons, Ltd.

Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose.

BackgroundKojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production.ResultsA kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose.ConclusionsKojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.

Feeding Deterrence, Acute Toxicity and Sublethal Growth Effects of Kojic Acid Isolated from Aspergillus funiculosus

Aspergillus funiculosus NCIM 1029 has been reported for the production of kojic acid exhibiting antibacterial, antifungal and larvicidal activity against 3rd instar larvae of Aedes aegypti. Aim of the Study: To check the antifeedant, acute toxicity and sublethal growth effects of fungal metabolite, kojic acid against the agriculture pest, Spodoptera litura. Materials and Methods: Kojic acid was checked for feeding deterrence activity against 4th instar larvae of S. litura in comparison to insect antifeedant, azadirachtin. Reduction in the weight and length of the larvae was evaluated after three days of exposure to kojic acid (400μg/ml). The effect of kojic acid on pupation and adult emergence was also studied. Results: On determination of feeding deterrence, the DC50 value for kojic acid was found to be 180.645 ± 6.35μg/ml on comparison to azadirachtin (DC50 = 41.372 ± 2.21). The LD50 & LD90 values of kojic acid were found to be 693.52± 51.31 and 1889.13 ± 279.03μg/ml, respectively. The reduction in larval weight and length were 83.24 and 87.50%, respectively. The percentage pupation and adult emergence were reduced by 69% and 64% for the exposed larvae. Conclusions: Kojic acid showed moderate feeding deterrence, acute toxicity and sublethal growth effects against S. litura. Keywords: Aspergillus funiculosus, kojic acid, Spodoptera litura, acute toxicity, antifeedant.

The non-metabolizable glucose analog D-glucal inhibits aflatoxin biosynthesis and promotes kojic acid production in Aspergillus flavus.

BackgroundAflatoxins (AFs) are potent carcinogenic compounds produced by several Aspergillus species, which pose serious threats to human health. As sugar is a preferred carbohydrate source for AF production, we examined the possibility of using sugar analogs to inhibit AF biosynthesis.ResultsWe showed that although D-glucal cannot be utilized by A. flavus as the sole carbohydrate source, it inhibited AF biosynthesis and promoted kojic acid production without affecting mycelial growth when applied to a glucose-containing medium. The inhibition occurred before the production of the first stable intermediate, norsolorinic acid, suggesting a complete inhibition of the AF biosynthetic pathway. Further studies showed that exogenous D-glucal in culture led to reduced accumulation of tricarboxylic acid (TCA) cycle intermediates and reduced glucose consumption, indicating that glycolysis is inhibited. Expression analyses revealed that D-glucal suppressed the expression of AF biosynthetic genes but promoted the expression of kojic acid biosynthetic genes.ConclusionsD-glucal as a non-metabolizable glucose analog inhibits the AF biosynthesis pathway by suppressing the expression of AF biosynthetic genes. The inhibition may occur either directly through interfering with glycolysis, or indirectly through reduced oxidative stresses from kojic acid biosynthesis.

Kojic Acid production from agro-industrial by-products using fungi.

A total of 278 different isolates of filamentous fungi were screened using synthetic medium for respective ability to produce kojic acid. Nineteen, six, and five isolates proved to be low, moderate, and high kojic acid producers, respectively. Levels of kojic acid produced were generally increased when shaking cultivation was used rather than those obtained using static cultivation. A trial for the utilization of 15 agro-industrial wastes or by-products for kojic acid production by the five selected higher kojic acid producer isolates was made. The best by-product medium recorded was molasses for kojic acid. A. flavus numbers 7 and 24 were able to grow and produce kojic acid on only 12 out of 15 wastes or by-products media. The best medium used for kojic acid production by A. flavus number 7 was rice fragments followed by molasses, while the best medium used for kojic acid production by A. flavus number 24 was the molasses followed by orange, pea, and rice fragments. An attempt for production of kojic acid using a 1.5 L laboratory fermentor has been made. Aspergillus flavus number 7 was used and grown on molasses medium; maximum level (53.5 g/L) of kojic acid was obtained after eight days of incubation.

Production Technology and Applications of Kojic Acid

Kojic acid is produced industrially by Aspergillus species in aerobic fermentation. The production of kojic acid is increasing because of its commercial value in industry. Kojic acid has various applications in several fields. It is widely used in cosmetic industry, medicine, food industry, agriculture and chemical industry. Nowadays, kojic acid plays a crucial role in cosmetic, especially skin care products because it can enhance the ability to prevent exposure to UV-radiation. Kojic acid continues to attract attention because of its economic potential in medical field as an anti-inflammatory drug and painkiller. In food industry, kojic acid is used in post harvest process as an anti-speck and an anti-browning agent for agricultural product. Due to various usage of this organic molecule, the demand of kojic acid has been increasing rapidly. Thus the studies to improve the kojic acid production are still extensively conducted.

Improvement of kojic acid production in Aspergillus oryzae B008 mutant strain and its uses in fermentation of concentrated corn stalk hydrolysate

A strain designated M866, producing kojic acid with a high yield, was obtained by combining induced mutation using ion beam implantation and ethyl methane sulfonate treatment of a wild type strain of Aspergillus oryzae B008. The amount of kojic acid produced by the strain M866 in a shaking flask was 40.2 g/L from 100 g/L of glucose, which was 1.7 times higher than that produced by wild strain (23.58 g/L). When the mixture of glucose and xylose was used as carbon source, the resulting kojic acid production was raised with the increasing of glucose ratios in the mixture. With concentrations of glucose at 75 g/L and xylose at 25 g/L mixed in the medium, the production of kojic acid reached 90.8 %, which was slightly lower than with glucose as the sole source of carbon. In addition, the kojic acid fermentation of the concentrated hydrolysate from corn stalk was also investigated in this study, the maximum concentration of kojic acid accumulated at the end of the fermentation was 33.1 g/L and this represents the yield based on reducing sugar consumed and the overall productivity of 0.36 g/g and 0.17 g/L/h, respectively.

Solid-State Bioconversion of Pineapple Residues into Kojic Acid by Aspergillus flavus: A Prospective Study

—Kojic acid is an organic acid that is widely used as an ingredient for dermatological products, precursor for flavor enhancer and also as anti-inflammatory drug. The present study was undertaken to test the feasibility of pineapple residues as substrate for kojic acid production by Aspergillus flavus Link 44-1 via solid-state fermentation. The effect of initial moisture content, pH and incubation time on kojic acid fermentation was investigated. The best initial moisture content for kojic acid production from pineapple residues was observed at 70% (v/w) whereas initial culture pH 2.5 was identified to give high production of kojic acid. The optimal range of incubation time was identified between 8 and 14 days of incubation which corresponded to highest range of kojic acid produced. The results from this study pronounce the promising usability of pineapple residues as alternative substrate for kojic acid production by A. flavus Link 44-1.

To Optimise the Cultural Conditions on the Production of Kojic Acid byAspergillusSpecies

The objective of the present study was to optimise the cultural conditions on the production of Kojic acid by Aspergillus species, namely, Aspergillus flavus, A. niger, A. parasiticus and A. ochraceous. Four different fungal strains of Aspergillus were subjected to different culture media, pH, temperature, carbon sources, nitrogen sources and salt concentration. The study concluded that out of four fungal strain A. parasiticus in yeast extract sucrose media along with wheat extract produced maximum kojic acid in static condition. Maximum kojic acid was produced on 11th day at pH 3.0, temperature 18°C where xylose (0.25 g/25 ml of media) was found to be best carbon source, lysine (0.005 g/25 ml of media) was found to be best nitrogen source and MgSO4 (0.005 g/25 ml of media) was found to be best salt source.

Aspergillus oryzae laeARegulates Kojic Acid Synthesis Genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.

Loss of msnA, a Putative Stress Regulatory Gene, in Aspergillus parasiticus and Aspergillus flavus Increased Production of Conidia, Aflatoxins and Kojic Acid

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.

Kojic acid production from glycerol: Optimization using central composite rotatable design

experiments, the biosurfactant stimulated IL-1 , IL-12, IL-18 and TNFproduction of humanmonocytes and displayed no cytotoxicity against human lymphocytes. Recently developed optimization procedures for the biosurfactant synthesis and recovery would broaden its potential applications in new advanced technologies, such as environmental bioremediation, improved polymeric material construction and biomedicine. Acknowledgements: Research was funded by the Russian Academy of Sciences (MCB Program) and the Federal Target Program “Scientific and Science-Pedagogical Personnel for Innovative Russia” for 2009–2013 years.

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae

Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism.

Dioctatin A is a strong inhibitor of aflatoxin production by Aspergillus parasiticus

Dioctatin A (DotA), a metabolite of Streptomyces, is known to be an inhibitor of human dipeptidyl aminopeptidase II. Here, it was found that DotA strongly inhibited aflatoxin production by Aspergillus parasiticus, with an IC50 value of 4.0 microM. The mycelial growth of the fungus was not affected by the addition of DotA at a concentration of 50 microM, but inhibition of conidiation was observed at the same concentration. DotA inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, and it strongly reduced the mRNA levels of genes encoding aflatoxin biosynthetic enzymes, and significantly decreased the mRNA level of aflR, which encodes a key regulatory protein for aflatoxin biosynthesis. In addition to these genes, the mRNA level of brlA, which encodes a conidiation-specific transcription factor, was also reduced by the addition of DotA. It was also found that DotA dramatically enhanced kojic acid production by the fungus. Furthermore, DotA inhibited production of sterigmatocystin, which is a toxic aflatoxin biosynthetic intermediate, and it also inhibited conidiation in Aspergillus nidulans. These results indicate that DotA has pleiotropic effects on regulatory mechanisms of fungal secondary metabolite production and differentiation, leading to inhibition of aflatoxin production.

Biotransformation of various carbon sources to kojic acid by cell-bound enzyme system of A. flavus Link 44-1

The ability of cell-bound enzyme of Aspergillus flavus Link 44-1 for production of kojic acid was studied in resuspended cell system. Cell material was produced in batch fermentation using 2 L stirred tank fermenter. The cell mycelia were then resuspended into 250 mL shake flask containing various carbon sources solution. Among the carbon sources tested, glucose gave the highest kojic acid yield based on carbon consumed (0.365 g/g) followed by sucrose (0.279 g/g), starch hydrolysate (0.212 g/g) and fructose (0.195 g/g). The rate of biotransformation was increased with increasing mycelial cell. Kojic acid production was also varied with different glucose and sucrose concentrations. The highest production was obtained at 100 g/L glucose and 100 g/L sucrose with a final kojic acid concentration of 45.3 and 33.4 g/L, respectively. The rate of biotransformation of glucose and sucrose to kojic acid followed the Michaelis–Menten equation, suggesting that the biotransformation rate vary with substrate concentration similar to the behaviour of many enzymes reaction.

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 gL(-1)day(-1) after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 gL(-1)day(-1), a productivity of 5 gL(-1)day(-1) was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.

Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production.

By combining induced mutation, using NTG and UV irradiation, and protoplasting of a wild type strain of Aspergillus oryzae ATCC 22788, a hyper-producing strain was obtained that accumulated 41 g kojic acid l(-1) in shake-flasks, which was 100-fold higher than that in the wild type strains. Similar production of kojic acid was obtained in 5 l stirred-tank fermentations.

Importance of carbon source feeding and pH control strategies for maximum kojic acid production from sago starch by Aspergillus flavus

To maximize kojic acid production by Aspergillus flavus Link 44-1 using gelatinized sago starch as a carbon source, the performance of different fermentation modes (batch and fed-batch with different feeding modes) was investigated in an 8- l stirred tank fermentor. The addition of a large volume of concentrated sago starch (140 g/ l) 2 d after the start with an initial starch concentration of 60 g/ l produced the maximum concentration of kojic acid (16.43 g/ l), which was about 4 times higher than that for batch fermentation of 100 g/ l sago starch. Further improvement of kojic acid production was obtained by adding small amounts of concentrated starch (140 g/ l) intermittently at 2-d intervals to the culture. Using this technique of fermentation, the dissolved oxygen tension (DOT) can be controlled at high levels (40–50% saturation) during the active growth phase, which is required for the enhanced secretion of α-amylase used for saccharification of starch and also for the production of mycelia with higher ability in synthesizing kojic acid. Maintaining the culture pH at 3 throughout the intermittent fed-batch fermentation reduced kojic acid production (7.26 g/ l) significantly. Very little kojic acid (0.93 g/ l) was produced when the pH was kept at 4 during the growth phase and then lowered to 3 during the production phase. A high level of kojic acid production (31.00 g/ l) was achieved in fermentation where the pH was not controlled throughout the cultivation or not controlled during the growth phase but kept at 3 during the production phase. This result indicates that the pH control strategy is important; with the variation in culture pH during the growth phase being most significant and critical to kojic acid production.