Optimization of production and evaluation of microbial kojic acid obtained from sugarcane molasses (SCM) by Aspergillus sp.

Толық мәтін

1. Introduction

Sugars, fibers, proteins, and minerals are the main ingredients of the goods of agricultural or animal origin that are produced by industrial processing. These agro-industrial wastes are mostly composed of lignocellulosic components, which include cellulose, hemicelluloses, and lignin [1]. Low-cost agro-industrial sources such as whey, sugarcane bagasse, maize steep liquor, wheat bran, soy bean meal, and others have been used in place of synthetic nitrogen and carbohydrate sources [2]. To make such wastes more palatable to microorganisms, they can undergo a range of physical, chemical, and enzymatic processing [3].

Organic acids can be manufactured industrially with the use of fermentative processes and variety of microorganisms. It is known that some Aspergillus fungi produce high concentrations of several organic acids, including citric acid, gluconic acid, and so on. However, some organic acids such as lactic acid, gibberellic acid, malic acid, and kojic acid are formed in trace amounts [4]. These acids are very advantageous for scientific, medical, and economic applications. In 1907, Saito used steamed rice to produce Aspergillus oryzae and isolated the kojic acid from the mycelial mat. Eventually, the rice was referred to as Koji, and the phrase Kojic acid was first used in 1913 by a scientist by the name of Yabuta [5].

Numerous research had proposed using agro-industrial wastes or byproducts for the fungal production of beneficial products, the waste or byproducts included cheese whey, sugar cane molasses, fruits, vegetables, and maize steep liquor [3, 6].

Kojic acid (5-hydroxy-2-hydroxymethylgamma-pyrone, or KA) is a significant secondary metabolite that can be generated from carbohydrates using a variety of carbon and nitrogen sources [7]. Using agricultural wastes and aerobic fermentation techniques, this task can be accomplished. Kojic acid is produced by Aspergillus species that are predominantly found in the section of flavi, namely Aspergillus flavus, Aspergillus oryzae, Aspergillus oryzae var effusus, Aspergillus tamari, and Aspergillus parasiticus, in addition to Penicillium sp. and certain bacteria [3]. Kojic acid, which has a melting point within the range of 151–154 °C, crystallizes as colorless prismatic needles in water when cooled by centrifugation [8].

According to Rasmey and Abdel-Kareem [9], it is more soluble in water, ethanol, and ethyl acetate and less soluble in ether and chloroform. Kojic acid has a wide range of applications in chemistry, medicine, food and cosmetics, even though it has been on the market for 40 years, research on the microbial production and application of kojic acid is still ongoing. It also has antibacterial, anticoagulant and chelating properties [10]. The main objectives of this work were to identify the Aspergillus species that can manufacture kojic acid (KA) using glucose and agro-industrial waste media, as well as to optimize the conditions of kojic acid synthesis.

2. Materials and methods

2.1. Materials

2.1.1. Microorganisms and chemicals

Aspergillus oryzae (AUMC.64), Aspergillus tamari (AUMC.43), Aspergillus niger (AUMC.42), Aspergillus fumigatus (AUMC.13602), Aspergillus nidulans (AUMC .13902), and Aspergillus japonicas (AUMC.14380) used in this work were obtained from Assiut University Moubasher Mycological Centre (AUMMC, Assiut, Egypt). Organisms were cultivated on potato dextrose agar slants (PDA) and kept at 4 °C.

The mold was inoculated on malt extract agar slants and incubated for five days at 30 °C in order to create spores. After incubation, the spores were taken out and re-injected into 50 ml of saline solution containing 0.1% of tween 80. The spores (5 × 10⁸ spores per ml^-1) were counted under a microscope, stored at 4 °C, and utilized as a stock inoculum. The chemicals were obtained from Sigma Chemical Company (St. Louis, Mo. USA).

2.1.2. Screening medium and culture conditions

The medium for kojic acid production consists of: (gl^-1) glucose, 100; yeast extract, 5; KH₂PO₄, 1.5; and MgSO₄, 0.5 combined at pH 4. 50 ml of the synthetic medium was added to 250 ml Erlenmeyer flasks, which were used to grow the experimental cultures. After being sterilized for 15 minutes at 121 °C, one milliliter of inoculum spore suspension (5 × 10⁸ spores per ml^-1) was added to the flasks after cooling. Then the cultures were incubated for ten days at 28 ± 2 °C.

2.1.3. Agro-industrial wastes

Various samples of agro-industrial wastes sugarcane molasses (SCM) were purchased from Sugars and Integrated Industries Egyptian Distillation Plants in Hawamdeia city, Giza, Egypt. Unsalted cheese whey as a by-product precipitated from milk during the production of cheese samples was kindly provided by the Dairy Department, Faculty of Agricultural-Cairo University. Also, the potato washing water resulting from the manufacture of chips was used in the fermentation process.

2.2. Methods

2.2.1. Screening of Aspergillus sp. for kojic acid production

The ability of the following fungal strains to produce kojic acid from glucose as the only carbon source in a liquid synthetic medium under aerobic conditions was tested: Aspergillus oryzae (AUMC.64), Aspergillus tamari (AUMC.43), Aspergillus niger (AUMC.42), Aspergillus fumigatus (AUMC.13602), Aspergillus nidulans (AUMC.13902), and Aspergillus japonicas (AUMC.14380) were tested. Since glucose is the most basic sugar, it was utilized to screen for Aspergillus species that produce kojic acid [8].

2.2.2. Treatment of sugarcane molasses

Molasses from sugarcane was kept at 4 °C. To create treated molasses, the molasses was diluted 1:1 with distilled water, and concentrated sulfuric acid was added to bring the diluted molasses’ pH to 4. After being heated to approximately 80 °C in a water bath for 1.5 hours, the molasses was centrifuged for 10 minutes at 4,000 rpm to precipitate the sludge while cooling. After filtering the treated molasses, the supernatant was moved to the fermentation flasks [11].

2.2.3. Screening of agro-industrial wastes for kojic acid production

Processed sugar cane molasses, whey, and potato water were used as fermentation media to produce kojic acid without adding any growth supplements. The previous media were sterilized at 121 °C for 15 minutes, followed by inoculation with tested Aspergillus strains and incubation at 28 ± 2 °C for 10 days. The ability of each experiment to yield kojic acid was evaluated [12].

2.2.4. Chemical composition of agro-industrial wastes

The chemical composition of agro-industrial wastes (sugarcane molasses, unsalted cheese whey, and potato water) were evaluated. Total carbohydrates were determined by the phenol sulfuric acid method [13]. Reducing the content of sugar was determined by DNSA method Utekar et al. [14]. Protein was also estimated using the Bradford method [15].

2.2.5. Determination of kojic acid content

According to Bentley, the amount of formed kojic acid in the supernatant culture was measured using a colorimetric technique with ferric chloride (FeCl₃) reagent. To prepare the reagent, one gram of FeCl₃.6H₂O was dissolved in 100 milliliters of 0.1 N HCl. The functional groups of the hydroxyl and pyrone rings in the samples react with the reagent to produce a deep red color. A spectrophotometer was used to measure the mixture’s absorbance at 500 nm. The kojic acid standard curve was used to calculate the kojic acid equivalent [9].

2.2.6. Fast detection of kojic acid

Screening the kojic acid producer was done fast (qualitatively), and the fungal-grown PDA plates were covered with a ferric chloride solution. The appearance of a deep red color during this procedure indicates the presence of kojic acid produced by the strain [10].

2.2.7. Fourier-transform infrared spectroscopy (FTIR)

For the further detection of the presence of kojic acid in extracted crystals, a Fourier-transform infrared spectroscope (FTIR) was used. The samples were analyzed on a Nicolet 380 FTIR using the KBr disk technique. Resolution was 4, and 16 scans were accumulated for each spectrum in a range of 500~4000 cm^-1, IR was used as a source and the detector is DTGS KBr. NICOLET 380 FT-IR, Thermo Scientific, made in China [16].

2.2.8. High performance liquid chromatography (HPLC)

To determine the KA content HPLC, Smart line, Knauer, Germany, was used, as well as a Kinetex XB-C18 Column (150 mm × 4.6 mm (Phenomenex®, USA)), operated at 40 °C. The injected volume was 20 µl in mobile phase of 0.1% acetic acid aqueous solution. Methanol 90:10 (v/v) Detection: VWD detector set at 270 nm was used. VWD: Variable Wavelength Detector [17].

2.2.9. Determination of dry weight

After the fermentation period ended, the biomass was recovered by filtering it through Whatman filter paper (No. 113), washed three times with distilled water, and dried for an entire night at 70 °C to determine its dry mass (DM) [18].

2.2.10. Optimization production conditions of kojic acid from sugarcane molasses

The study examined the effects of varying cultivating conditions on the biosynthesis of kojic acid, such as molasses concentration, initial pH value, and incubation period. Kojic acid was assayed to determine the impact of varying of the initial pH values (3.0, 4.0, 5.0, 6.0, and 7), varying molasses concentrations (3 to 10% with a 1% interval) and varying fermentation times (7 to 21 days) at the conclusion of each parameter [9].

2.2.11. Kojic acid extraction and crystallization

The process of extracting Kojic acid crystal involved filtering off the culture media using Wattman filter paper No. 1. The remaining solution was treated with ethyl acetate and refrigerated for 24 hours at 5 °C, causing the KA crystals to precipitate. The crystal was processed and dried at 80 °C for an hour the following day [10].

2.2.12. Determination of kojic acid antibacterial activity

Kojic acid’s antimicrobial activity was assessed using the well diffusion assay. Every 24 hour test strains of microorganisms with 1.0 × 10⁷ CFU ml^-1 was spread plated in 0.1 ml volume on the plate count agar, PCA medium (Oxoid, England). Subsequently, 50 μl of Kojic acid was added to each well. The plates were then incubated for 24 hour at 37 °C, and the inhibition of test strains of microorganisms was measured as the inhibition zone in millimeters [19].

2.2.13. Determination of kojic acid antioxidant activity

The degree of KA antioxidant activity is its capacity to scavenge 1.1-diphenyl-2-picrylhydrazyl (DPPH). Radical scavenging (%) = [(A0 – A1/A0) × 100] is the formula used by [20, 21, 22] to determine the percentage of radical scavenging activity. The absorbance of the sample extracts is A1, and the absorbance in the control sample is A0 [23].

2.2.14. Statistical analysis

The XLSTAT software version 2014, 5.03 (Addinsoft, New York, NY, USA) was used to analyze the experimental results using analysis of variance (ANOVA) in three repeats. The results were expressed as the mean ± standard error of the mean. When calculating the significance of mean differences between samples, a p-value of less than 0.05 was deemed as significant.

3. Results and discussion

3.1. Screening of Aspergillus sp. for kojic acid production

The ability of the Aspergillus species A. oryzae, A. tamari, A. niger, A. fumigatus, A. nidulans, and A. japonicas, to produce kojic acid was tested, and the results are recorded in the Table 1. The obtained results demonstrate that after ten days of cultivation, the various strains’ abilities to produce kojic acid varied. The highest kojic acid production was recorded for A. oryzae AUMC64 (12.599 g. L^-1) followed by A. tamari AUMC43 (7.003 g. L^-1), A. nidulans AUMC13902 (1.527 g. L^-1), A. fumigatus AUMC13602 (0.694 g. L^-1), and A. japonicas AUMC14380 (0.276 g. L^-1), while the lowest value was for niger AUMC42 (0.115 g. L^-1). Therefore, A. oryzae AUMC64 and A. tamari AUMC43 were chosen for additional testing (Table 1). The results show a high correlation (r = 0.97) between the two approaches when it comes to the relation between the qualitative and quantitative methods. These results are consistent with those of [9, 11, 24]. These variations in the biomass and kojic acid production can be attributed to species differences or culture conditions. Aspergillus oryzae was a great starting strain for the safe synthesis of kojic acid because it has been proven in several studies to have low pathogenicity and to not produce aflatoxins or any other carcinogenic metabolites. Aspergillus species can produce the amylase enzyme, which hydrolyses starch into simple sugars that can then be converted into kojic acid, according to research by [25]. The type of fermentable sugars present in the culture medium determines the amount of kojic acid being produced (g. L^-1).

 

Table 1. Selection and screening of various Aspergillus sp. for kojic acid production

Таблица 1. Отбор и анализ различных видов Aspergillus sp., используемых для производства койевой кислоты

Strains	Kojic acid production (g. L-1)	Red zone (mm)
Aspergillus nidulans AUMC13902	1.527 c ± 0.03	3.47c ± 0.06
Aspergillus oryzae AUMC64	12.599a ± 0.06	4.97a ± 0.05
Aspergillus fumigatus AUMC13602	0.694d ± 0.02	2.13d ± 0.05
Aspergillus japonicas AUMC14380	0.276e ± 0.04	2.01d ± 0.09
Aspergillus niger AUMC42	0.115f ± 0.07	1.03e ± 0.01
Aspergillus tamari AUMC43	7.003b ± 0.04	4.07b ± 0.06

The experimental values (means and SD for n = 3) with small letter are significantly different (P ≤ 0.05).

 

3.2. Chemical composition of agro-industrial waste

The chemical composition of agro-industrial wastes (sugarcane molasses, unsalted cheese whey, and potato water) was evaluated. Total content of carbohydrates was determined, and the results were as follows: 56.01, 12, and 20% for molasses, whey, and potato water, respectively. Meanwhile, reduction sugar was found to be 24.90, 4, and 0.2% for molasses, whey, and potato water, respectively. Protein content was also estimated, and the results were as follows: 5, 2, and 0% for molasses, whey, and potato water, respectively.

3.3. Screening of the agro-industrial waste for kojic acid production

Various samples of agro-industrial wastes (cheese whey, potato water, and sugarcane molasses) were tested as a carbon source for the growth of A. oryzae, and A. tamari and as a natural medium for kojic acid production. The results are shown in the Table 2. The results indicated that the highest productivity of kojic acid was obtained from SCM (14.28 g. L^-1), followed by cheese whey and potato water (5.34 g. L^-1 and 0.55 g. L^-1, respectively). These results were in harmony with those previously obtained by [3, 11]. Egyptian SCM has detectable levels of several vitamins, including riboflavin and thiamin, and roughly 52% total sugar (glucose, sucrose, and fructose), as well as 0.46% total nitrogen. These substances may encourage the synthesis of kojic acid in each type of molasses [12].

 

Table 2. Screening of the agro-industrial waste for kojic acid production (g. L^-1) by A. oryzae and A. tamari

Таблица 2. Анализ агропромышленных отходов, используемых для производства койевой кислоты (g. L^-1) при помощи A. oryzae и A. tamari

Agro-industrial waste	Aspergillus oryzae AUMC64		Aspergillus tamari AUMC43
	Kojic acid production (g. L-1)	Dry weight (g. L-1)	Kojic acid production (g. L-1)	Dry weight (g. L-1)
Synthetic media	12.59b ± 0.01	41.9b ± 0.07	7.01b ± 0.06	38.3b ± 0.31
Potato water	0.55d ± 0.03	7.9d ± 0.49	0.66d ± 0.04	5.7d ± 0.42
Cheese whey	5.34c ± 0.02	24.9c ± 0.34	4.93c ± 0.03	25.0c ± 0.40
Sugarcane molasses	14.28a ± 0.04	52.7a ± 0.36	9.61a ± 0.06	50.3a ± 0.86

The experimental values (means and SD for n = 3) with small letter are significantly different (P ≤ 0.05).

 

3.4. Optimization of kojic acid production in SCM media by A. oryzae, and A. tamari

3.4.1. Effect of duration of fermentation period on kojic acid production

The effects of the duration of fermentation period on the production of kojic acid from SCM by A. oryzae, and A. tamari were investigated. The data presented in the Table 3 indicate that the production of kojic acid increased gradually along with the duration of the incubation period, reaching its maximum value (25.9 g. L^-1) at 10 days before declining. These outcomes corroborated those previously attained by [12, 26]. According to Rasmey et al. [11], the mycelium’s breakdown of kojic acid into oxalic and acetic acid when glucose levels are low may be the cause of the decrease in kojic acid that occurs with longer period of incubation.

 

Table 3. Effect of various fermentation periods on yield of kojic acid being produced from SCM by A. oryzae, and A. tamari

Таблица 3. Влияние различных периодов ферментации на объем койевой кислоты, получаемой из ПСТ при помощи A. oryzae и A. tamari

Days	Aspergillus oryzae AUMC64		Aspergillus tamari AUMC43
	Kojic acid production (g. L-1)	Dry weight (g. L-1)	Kojic acid production (g. L-1)	Dry weight (g. L-1)
3	10.22d ± 0.06	8.7g ± 0.06	8.19d ± 0.07	2.7g ± 0.06
7	21.73b ± 0.09	22.4f ± 0.01	15.37b ± 0.09	8.3f ± 0.12
10	25.91a ± 0.06	26.3e ± 0.06	18.04a ± 0.08	15.3e ± 0.12
13	20.02c ± 0.09	22.7d ± 0.12	13.90c ± 0.09	21.0d ± 0.10
15	15.71e ± 0.07	27.7c ± 0.06	10.70e ± 0.06	25.7c ± 0.06
18	13.20f ± 0.08	32.7b ± 0.06	9.92f ± 0.05	36.3b ± 0.06
21	10.90g ± 0.01	35.7a ± 0.06	7.04g ± 0.08	42.3a ± 0.06

The experimental values (means and SD for n = 3) with small letter are significantly different (P ≤ 0.05).

 

3.4.2. Effect of SCM concentrations on kojic acid production

The Table 4 presents the results of a test conducted to determine the effect of SCM concentrations in the medium on the production of kojic acid by A. oryzae and A. tamari. The outcome demonstrated that the production of kojic acid increased as molasses sugar concentrations rose, peaking at 10% sugar content and being produced by A. oryzae, and A. tamari (25.9–18.9 g. L^-1). The production of the acid decreased as the concentration of sugar molasses (15–20–25%) was increased further. Fermentation rates decreased as a result of both decreased water activity and the beginning of plasmolysis. Elevating the initial concentration of sugar led to a noteworthy rise in residual sugar content, potentially resulting from the microorganisms’ incapacity to metabolize elevated sugar levels. It was found that the osmotic pressure had a negative impact because the acid production abruptly stopped. These outcomes concurred with the preliminary findings of [11]. Once the fungal growth reached the stationary phase, the synthesis of kojic acid ceased when the certain concentration of medium’s glucose was reached. According to studies by [25, 26], the synthesis of kojic acid was impacted by an excessive concentration of carbon sources.

 

Table 4. Effect of various concentrations of sugarcane molasses on the yield of kojic acid being produced by A. oryzae and A. tamari

Таблица 4. Влияние различных концентраций патоки сахарного тростника на объем койевой кислоты, получаемой при помощи A. oryzae и A. tamari

SCM concentration% (V/V)	Aspergillus oryzae AUMC64		Aspergillus tamari AUMC43
	Kojic acid production (g. L-1)	Dry weight (g. L-1)	Kojic acid production (g. L-1)	Dry weight (g. L-1)
5%	16.70d ± 0.01	14.2e ± 0.1	10.90d ± 0.01	12.7e ± 0.38
10%	25.90a ± 0.05	26.3d ± 0.12	18.91a ± 0.07	23.7d ± 0.21
15%	23.13b ± 0.05	32.3c ± 0.16	17.92b ± 0.04	31.3c ± 0.35
20%	17.59c ± 0.06	45.3b ± 0.25	12.58c ± 0.05	44.0b ± 0.26
25%	10.19e ± 0.05	52.7a ± 0.12	8.03e ± 0.07	53.7a ± 0.31

The experimental values (means and SD for n = 3) with small letter are significantly different (P ≤ 0.05).

 

3.4.3. Effect of pH on production of kojic acid

The Table 5 presents the results of a study conducted to examine the effect of the initial pH value in the molasses fermentation medium on the production of kojic acid from SCM by A. oryzae, and A. tamari The tested strains produced kojic acid at the best level at a pH of 4.0. Beyond pH 4.0, kojic acid production started to decline. Based on the initial pH of culture, the majority of research on how culture pH affects kojic acid production and growth was done. A. oryzae, and A. tamari are two examples of fungi that can produce kojic acid within the pH range of 3 to 7. High pH had a major impact on the metabolism of fungi. The fungus’s metabolism shifted to other pathways rather than producing kojic acid. Thus, a high pH further decreased the yield of kojic acid [27]. At pH 4.0, A. oryzae and A. tamari demonstrated the highest yield of crystals of kojic acid. The growth of the fungus and the yield of kojic acid decreased as the pH of the culture medium increased [10]. According to [9], the structure and function of enzymes were influenced by the pH of the culture medium because the enzymes are proteins with ionizable groups. Determining the perfect pH is crucial because it has a significant impact on the best way to produce the enzymes needed to obtain kojic acid.

 

Table 5. Effect of various initial pH values on production of kojic acid from SCM by A. Oryzae and A. tamari

Таблица 5. Влияние различных начальных значений pH на объем койевой кислоты, получаемой из ПСТ при помощи A. oryzae и A. tamari

pH	Aspergillus oryzae AUMC64		Aspergillus tamari AUMC43
	Kojic acid production (g. L-1)	Dry weight (g. L-1)	Kojic acid production (g. L-1)	Dry weight (g. L-1)
3	19.65c ± 0.05	15.7b ± 0.04	14.30c ± 0.04	16.0b ± 0.30
4	25.91a ± 0.05	26.8a ± 0.25	18.95a ± 0.09	23.7a ± 0.50
5	21.65b ± 0.06	15.6b ± 0.05	15.91b ± 0.08	17.3b ± 0.15
6	18.12d ± 0.07	11.5c ± 0.31	11.21d ± 0.01	13.3c ± 0.15
7	15.04e ± 0.05	7.9d ± 0.26	8.31e ± 0.06	8.3d ± 0.06

The experimental values (means and SD for n = 3) with small letter are significantly different (P ≤ 0.05).

 

3.5. Production of kojic acid in optimal conditions

According to this study, 10% concentration of SCM provided the best conditions for the fermentation of A. oryzae AUMC64 and A. tamari AUMC43, which produce kojic acid. In perfect environmental conditions, the pH and incubation period were 4.0 and 10 days, respectively. These outcomes are exactly the same as those noted by multiple researchers [9, 10, 12, 27].

3.6. Extraction and characterization of produced kojic acid

The process of extracting kojic acid crystals involved filtering culture media using Wattman filter paper No. 1. As previously mentioned, the left mycelial mass was also weighted according to the procedures to determine wet and dry weight. After being treated with ethyl acetate, the remaining solution was kept in a refrigerator at 5 °C for a full day. The following day, crystals of KA were visible at the beaker’s bottom. To o btain such crystals, filtration was done, and they were then oven dried for one hour at 80 °C [10].

3.6.1. Infrared spectroscopy via Fourier-transform infrared spectroscopy (FTIR)

The sample of kojic acid’s FTIR spectrum displays peak wave number values peculiar for the functional groups that are comparable to those of regular kojic acid (Figure 1). Functional groups 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 á -disubstituted ring) are among the functional groups where the bands are present [17].

 

Figure 1. Peak wave number values of FTIR for standard kojic acid

Рисунок 1. Значения пиковых волновых чисел по ИК-Фурье спектроскопии у стандартной койевой кислоты

 

3.6.2. High performance liquid chromatography (HPLC)

The compound with the same retention time as the standard kojic acid was found among the best HPLC results for A. oryzae (Figure 2). All the experimental analysis (HPLC and FTIR) indicated that kojic acid was produced by both fungus [10].

 

Figure 2. HPLC of kojic acid obtained from A. oryzae (A) and A. tamari (B)

Рисунок 2. ВЭЖХ койевой кислоты, получаемой при помощи A. oryzae (A) и A. tamari (B)

 

3.6.3. Antibacterial activity of kojic acid

The antimicrobial activities of KA produced by A. oryzae AUMC64 and A. tamari AUMC43 against the selected test strains of microorganisms (Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Salmonella typhimurium) was investigated. Our results are presented in the Table 6. The maximum zone of inhibition (20–13.2 mm) was observed with the cultures, Escherichia coli and Salmonella typhimurium, followed by Staphylococcus aureus and Bacillus cereus (9.6–8.3 mm) for KA produced by A. oryzae AUMC64. Such a result indicates that these organisms were highly sensitive to antimicrobial compounds and kojic acid. These results agreed with those previously obtained by [5, 19].

 

Table 6. Antibacterial activities of kojic acid produced by A. oryzae and A. tamari

Таблица 6. Антибактериальная активность койевой кислоты, получаемой при помощи A.oryzae и A. tamari

Test strains of microorganisms	Kojic acid concentration (mg/ml)			Inhibition zone (mm)
	A. oryzae			A. tamari
	0.5	1	2	0.5	1	2
S. aureus	2.5	6.6	9.6	1.6	4.5	7.1
B. cereus	1.8	6.1	8.3	1.1	3.8	6.4
E. coli	4.2	8.4	20	2.5	5.7	13.2
Sal. typhimurium	3.2	7.3	11.1	1.9	5.1	7.6

 

3.6.4. Antioxidant activity of kojic acid

The antioxidant activity of KA produced by two fungal strains was tested by their scavenging effect on DPPH radicals under cultural conditions, and the results are recorded in the Figure 3. The results indicated that the antioxidant activity of KA extract produced by A. oryzae AUMC64 and A. tamari AUMC43 was 79.1 and 62.42%, respectively. These results indicate that the antioxidant percentage is correlated with amounts of KA in the culture medium. KA is a good chelator of transitional metal ions and a good scavenger of the free radical DPPH. Moreover, KA has potential activity in depigmentation processes through chelating the copper ion present in the active site of tyrosinase, which mediates the formation of melanin from the amino acid tyrosine [28]. The potential antioxidant activity of KA approves the benefits of its application in the food industry, where it has been used in the post-harvest process as an anti-speck and an anti-browning agent for the agricultural products [29].

 

Figure 3. Antioxidant activity of kojic acid produced by A. oryzae and A. tamari

Рисунок 3. Антиоксидантная активность койевой кислоты, получаемой при помощи A.oryzae и A.tamari

 

4. Conclusion

During the study of the possibility of utilizing some agro-industrial wastes to produce microbial kojic acid using both A. oryzae and A. tamari, the results demonstrated the possibility of using SCM as a fermentation medium and obtaining high level of kojic acid production compared to the synthetic medium. Fermentation conditions were also controlled to maximize the productivity. Some properties of kojic acid were studied, such as its effect as an antioxidant and antibacterial substance, and the results positively proved its ability to do this. In the future, we can continue to study the possibility of using waste for fermentation producing biological compounds and running works to reduce the pollution resulting from these wastes on the path to sustainable development.

Авторлар туралы

D. Mohamed

Cairo University

Хат алмасуға жауапты Автор.
Email: Dalia.header@agr.cu.edu.eg
ORCID iD: 0009-0004-3487-8759

Teaching Assistant, Department of Food Science, Faculty of Agriculture

Египет, Giza

A. Alian

Cairo University

Email: dr.alian@hotmail.com
ORCID iD: 0009-0003-1846-5375

Professor, Department of Food Science, Faculty of Agriculture

Египет, Giza

R. Mohamed

Cairo University

Email: reda_karrim@agr.cu.edu.eg
ORCID iD: 0000-0001-5817-9187

Associate Professor, Department of Food Science, Faculty of Agriculture

Египет, Giza

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