Layer 1: Convolution of 96 filters, size 7 x 7, stride 2, padding 3
2. Materials and methods 1. Materials
2.6. Evaluation of antibacterial activity of P. sanguineus extracts
The antibacterial activity of P. sanguineus was evaluated on 5 bacterial strains including Gram- negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella spp.) and Gram-positive (Bacillus cereus, Staphylococcus aureus) by measuring the zone of inhibition diameter via agar well diffusion method. After spreading the indicator culture on Luria-Bertani (LB) medium, the hole in the agar plate was made with a cork borer with a diameter of 5 mm. About the antibacterial solution, 100 mg of the mushroom extract was mixed thoroughly in 10 mL of distilled water to obtain a final concentration of 10 mg/mL which was then pipetted 10 µL into the holes. The positive control was the ampicillin antibiotic (0.05 mg/mL), the negative control was the sterilized distilled water. Formula to calculate antibacterial diameter: ΔD = D - d, where: ΔD is antibacterial diameter (mm), D is diameter of outer circle, d is diameter of agar hole (mm). Antibacterial efficiency (%) was calculated as: Hr (%) = ΔDs / ΔDp x100%, where: ΔDs is zone of inhibition diameter of the sample (mm), ΔDp is by the zone of inhibition diameter of the ampicillin positive control (0.05 mg/mL).
Data analysis
The experiments were repeated three times and the data of the experimental results were processed using Minitab 16 statistical software and MS Excel 2016.
3. Results and discussion
Fungal isolation and morphology identification
The collected mushroom samples were analyzed for anatomical morphology, spore structure, as well as biological characteristics.
As the result, the fruiting bodies are single or clustered on rotten trees, with short or inconspicuous stalks. The shape of the mushroom cluster connects the base and the mushroom cap, attached to the side. Mushroom fruit bodies are brilliant red-orange, the surface of the fruiting bodies is from light orange to dark orange (Figure 1). The mushroom cap part thrives, the edge of the mushroom on both sides of the stem can spread wide and spill over into contact with each other to form a canopy, which can connect many fruit body caps into a cluster of fruiting bodies. The stem of mushroom was measured and reported from 0.5 to 3 cm in length, from 3 to 10 mm in the diameter.
Figure 1. Fruiting body morphology of mushroom collected in Tra Co commune, Tan Phu district, Dong Nai province. A: The upper surface of the fruit body of mushroom sample; B: The
underside of the fruiting body of mushroom sample;
The mushroom cap was cut vertically consists of the following layers: the outer layer of the mushroom is thin, smooth and tough; the mushroom flesh has a uniform structure by very fine fibers, light orange in color, about 2 mm thick; the stratum cell layer is composed of a straight tubular layer. The filamentous system of the stratum corneum is straight, light red to dark orange, the length of the stratum receptor changes gradually from the inside of the fungal stalk to the edge of the cap, the average length is 1-1.5 mm, the size is about 128 - 150 µM and the pore density is about 4-6 holes/mm (Figure 2).
Figure 2. The stratum corneum on the underside of the cap of mushroom; A: Straight tube stage observed under the microscope 40X objective; B: Lower surface layer observed under
microscope 100X objective.
A B
A B
Figure 3. The spores observed under the microscope 100X objective (A), The germinating hole shrinks like a small protrusion; (B) The sporangium is a thin, almost transparent, bilayer.
Spores are cylindrical, slightly flattened, slightly curved (Figure 3). Spore diameter is about 4.1 - 6.2 m x 7.5 - 10.2 m, smooth. The outer shell of the spores is a thin, smooth, almost transparent bilayer. Germination hole small, clearly visible with a small protrusion at one end (Figure 3).
Samples of orange fungus suspected to be P. sanguineus after being collected at different locations were used to conduct biochemical tests. Mushroom caps turn black when exposed to KOH (5%).
After that, drying at 50oC for 1 h, the mushroom sample became greenish brown, thus giving rise to a xanthochromia-pseudoreaction.
After analyzing the mushroom anatomical morphology and spore structure and biological characteristics, based on the taxonomic description of Ryvarden & Johansen (1980), two samples of Pyc-1 and Pyc-2 fungi were identified belonging to the order Polyporales, family Polyporaceae, genus Pycnoporus, highly similar to P. sanguineus species. Comparing the macroscopic, microscopic, mycelial structure of Pyc-1 and Pyc-2, both mushroom samples are similar to the description of Pycnoporus sanguineus on the database of Mycobank. However, it is necessary to analyze the ITS rDNA sequence of the two fungal samples Pyc-1 and Pyc-2 to suggest the exact species.
Figure 4. Electrophoresis results of total DNA genome extract. M1, M2 are the Pyc-1 ITS, Pyc-2 ITS samples, respectively (A). Electrophoresis of PCR product amplified ITS sequence. (M) ladder 100 bp (Bioline); (-) negative control; M1, M2 are the Pyc-1 ITS, Pyc-2 ITS samples,
respectively (B).
A B
Total genomic DNA after extraction was checked by 1.5% agarose electrophoresis (Figure 4A).
The results of electrophoresis showed that the amount of total genomic DNA was of the required quality to conduct PCR amplification of the ITS rDNA gene fragment (Figure 4).
Primers ITS 5 (5′-GGAAGTAAAAGTCGTAACAAGG) and ITS 4 (5′- TCCTCCGCTTATTGATATGC) amplify the ITS I region, the 5.8S rDNA, the ITS II region, and part of the 28S rDNA region. After purification by QIAquick PCR Purification Kit (QIAGEN), the product of ITS amplifying gene fragment is Pyc-1 ITS, Pyc-2 ITS is electrophoresed for clear bands with the expected size from 600-800 bp (figure 4B).
ITS sequencing analysis, phylogenetic tree construction, and identification
The results of sequencing the ITS region of two mushroom samples obtained in Cu Chi (Pyc-1 ITS) and Dong Nai (Pyc-2 ITS) showed good signals, uniform peaks, and no overlap. All the above two sequences were checked using Chromaspro 1.34 software. As a result, two sequences of Pyc- 1 ITS with the length of 650 bp and Pyc-2 ITS of 657 bp were obtained and submitted to NCBI with access number were MZ305063 and MZ305070, respectively. Sequence after adjusting BLASTn on NCBI, to compare with available sequences on gene bank. The results of genetic similarity of two samples Pyc-1 ITS and Pyc-2 ITS with species published on NCBI are shown in Table 1.
The results of rDNA ITS sequence analysis showed that both Pyc-1 ITS and Pyc-2 ITS samples belonged to the species Pycnoporus sanguineus with the highest percentage of similarity at 99.69%
and 99.84%, respectively. The genus Pycnoporus is now described as consisting of four main species, distinguished on the basis of their morphology, namely P. sanguineus, P. cinnabarinus, P.
coccineus and P.puniceus (Ryvarden & Johansen, 1980). To further analyze the relationships between species in the same genus Pycnoporus, nine gene sequences coding for the rDNA ITS structure of Pycnoporus species were obtained from the NCBI database including Pycnoporus sanguineus CBS 358.63 (AF363759.1); Pycnoporus sanguineus C 030 (JN003682.1); Pycnoporus coccineus IUM 4093 (KP255841.1); Pycnoporus coccineus NW 580 (EU520116.1); Pycnoporus cinnabarinus SS3 (AF 363571); Pycnoporus cinnabarinus I-937 (AF363772.1); Pycnoporus puniceus MUCL 47087 (FJ234199.1); Pycnoporus puniceus BCC 26408 (J372685.1); Pycnoporus puniceus BCC 27595 (FJ372686.1) (Figure 5).
Table 1. The genetic similarity coefficient of the two samples Pyc-1 ITS and Pyc-2 ITS with species of the genus Pycnoporus published on NCBI
Sample NCBI access number
Species Query Coverage
(%)
Percentage Identity (%)
Reference access number Pyc-1 ITS
MZ305063 Pycnoporus sanguineus 100 99.69 JN164981.1
Pyc-2 ITS MZ305070 Pycnoporus sanguineus 100 99.84 JN164981.1
Figure 5. Phylogenetic tree showing the relationship of Pyc-1 and Pyc-2 fungal samples to other species of the genus Pycnoporus. The bootstrap number percentage is shown next to the branch
nodes. The ruler represents the number of nucleotides changed per site.
A 537 bp genomic region of the Pyc-1 ITS rDNA and Pyc-2 ITS rDNA was analyzed with the above 9 rDNA ITS sequences. Sequences were compared by Alignment analysis using ClustalW algorithm using MEGA X software. The phylogenetic tree was built using the Maximum Likelihood method using MEGA X software with a bootstrap index of 1000 according to the Tamura-Nei model. Analysis for 12 rDNA ITS sequences with out-group using rDNA ITS of Ganoderma lucidum ATCC 64251 (JQ520187.1). From the phylogenetic tree, it is easy to see that two samples of fungi, Pyc-1 and Pyc-2, belong to the group of P. sanguineus with a bootstrap index of 89, and have a close genetic relationship to P. coccineus (Figure 5). Although P. sanguineus and P. coccineus look very similar morphologically, but P. coccineus tends to prefer cold temperatures and can be found commonly in temperate regions, P. sanguineus is more commonly found in tropical regions. Based on BLAST results on NCBI database, phylogenetic tree analysis, it can be concluded that both mushroom samples collected from Cu Chi (Ho Chi Minh City) and Tan Phu (Dong Nai) are Pyc-1 and Pyc-2 belongs to the genus Pycnoporus, species Pycnoporus sanguineus.
After completing the identification, we selected Pyc-1 sample collected from Cu Chi to determine suitable growing conditions of the mushroom. From the fruiting body of Pyc-1 sample, the inner tissue was collected and isolated into a 10 cm diameter PDA petri dish. After 15 days, the milky white mycelium turned orange-red with age at the edge of the petri dish. The mycelium is thick in the center and thinner at the edge of the disc, growing evenly to form a concentric circle (Figure 6A and 6B).
Figure 6. Mycelium (Pyc-1) isolated on PDA petri dishes after 15 days of culture (A). Mycelium (Pyc-1) growing on secondary propagation medium after 21 days (B).
Optimization conditions for growing mycelium on rubber sawdust medium
The mycelium of P. sanguineus (Pyc-1) spread evenly to the substrate surface after 30 days. The mycelium of P. sanguineus was significantly different in the tested treatments. The results in Table 2 showed that the speed of fungal mycelium was fastest on the medium with an initial pH of 9 in the 10 days after inoculation (0.69 cm/day), and in the 20 days after inoculation (0.52 cm/day), and 30 days after inoculation (0.56 cm/day). This result showed that P. sanguineus was a suitable fungus for alkaline environments, consistent with the comments of Nguyen (2003) and Le (2001).
The characteristics of mycelium after 30 days of incubation in a dark room, in the pH 9 treatment, were strong mycelium, the mycelium part was white and thick, and the older mycelium gradually turned red-orange (not clear, should re-write). Therefore, bags of P. sanguineus fungal embryos in the treatment (pH 9) were suitable for experiments to investigate the formation of fungal fruiting bodies under different light conditions (Table 2).
The ability to spread mycelium versus initial pH was statistically analyzed by one-way ANOVA.
In each part of the day of cultivation, means that do not share a superscript letter are significantly different. The confidence level is 95%.
P. sanguineus fungus after 80 - 120 days cultured on rubber sawdust substrate at temperature 30oC
± 3oC, air humidity 80% - 90%, at different light levels. Fruiting bodies were collected after 75 - 90 days fruiting bodies formed and developed. As the results were shown in Table 3, the biological yield weight of P. sanguineus fungal fruiting bodies was affected under different light conditions.
Under 50% natural light (100 - 300 lux) conditions, the biological yield was the highest (8.86%) and the average dry weight was 20.39 g/bag. The formation of fruiting bodies of P. sanguineus was induced by light scattered light. However, under the light condition was totally dark (0 - 50 lux), the fruiting bodies could not develop normally and the biological yield was drastically reduced (3.18%).
A B
Mushroom weight and biological yield were statistical analyzed by one - way ANOVA. In each column, means that do not share a superscript letter are significant different. Confidence level is 95%.
Table 2. The speed of the mycelium of P. sanguineus (Pyc-1) depends on the pH conditions Time
(days) Initial
pH Ability to spread mycelium (cm)
Ability to spread mycelium spread by day
(cm/day)
10
7 5.21b 0.52
8 6.09ab 0.61
9 6.93a 0.69
10 5.5b 0.55
20
7 8.11b 0.41
8 9.48ab 0.47
9 10.48a 0.52
10 7.89b 0.39
30
7 12.24b 0.41
8 14.98a 0.50
9 16.83a 0.56
10 12.08b 0.40
Table 3. Biological yield of P. sanguineus fungal fruiting bodies depends on light intensity Covered light level Dried
substrate weight (g)
Fresh mushroom
weight (g)
Dried mushroom
weight (g)
Biological yield (%) Totally dark
(0 - 50 lux) 500 15.92b 7.32b 3.18b
50% natural light
(100 - 300 lux) 500 44.32a 20.39a 8.86a
70% natural light
(500 - 1000 lux 500 14.90b 6.86b 2.99b
100% natural light
(1500-2000 lux) 500 12.49b 5.75b 2.50b
Evaluation of antibacterial activity of extracts from P. sanguineus
The antibacterial activity of the extracts obtained from the fruiting bodies of P. sanguineus was investigated at the concentration of 10 mg/mL by the agar well diffusion method. The strains of bacterial were grown on plates, at the density of 106CFU/mL. At the agar holes appeared a zone
of inhibition against strains of E. coli, Salmonella spp., P. aeruginosa, S. aureus, B. cereus, no zone of inhibition was found against P. aeruginosa (Figure 7).
Figure 7. Antibacterial results of samples of P. sanguineus at the concentration 10 mg/mL on bacterial strains. A. E. coli ; B. Salmonella spp. ; C. S. aureus; D. B. cereus.
The highest antibacterial efficiency was the mushroom alcohol extract for E. coli was 96%
compared with the positive control, and the lowest was the mushroom water extract for Salmonella spp. was 46% compared to a positive control with the antibiotic ampicillin (0.05 mg/mL).
Table 4. Zone of inhibition of extracts from P. sanguineus (Pyc-1) fungi on tested bacterial strains at bacterial density 106 CFU/mL.
Bacterial Gram
Zone of inhibition ΔD (mm) Hr (%) Negative
control (-)
Positive control
(+) MWE MAE MWE MAE
E. coli - 0 14.80 10.80 14.20 73% 96%
P. aeruginosa - 0 12.50 ND ND ND ND
Salmonella spp. - 0 12.50 5.80 6.20 46% 50%
S. aureus + 0 14.40 9.80 11.20 68% 78%
B. cereus + 0 14.20 12.30 12.20 87% 86%
Control (-): Sterilized distilled water; Control (+): Ampicillin antibiotic (0.05 mg/mL); MWE:
mushroom water extract; MAE: mushroom alcohol extract. Hr (%): Resistance performance compared with positive control; ND: No inhibition zone
From the results of Table 4, the mushroom alcohol extract sample from P. sanguineus (Pyc-1) showed higher antibacterial ability than the mushroom water extract sample against E. coli, Salmonella spp., and S. aureus. The alcohol and water extracts inhibited the growth of B. cereus with similar antibacterial performance. Two samples of water extract and alcohol extract inhibited Gram-negative bacteria E. coli and Salmonella spp., as well as strains of Gram-positive bacteria S.
aureus and B. cereus, but not P. aeruginosa.
4. Conclusions
Mushroom samples Pyc-1 and Pyc-2 were isolated and identified with the scientific name Pycnoporus sanguineus. The growth rate of P. sanguineus mycelium (Pyc-1) was fastest on the medium with an initial pH level of 9 (0.59 ± 0.02 cm/day). The fruiting body of P. sanguineus fungus (Pyc-1) achieved the highest average dry weight of 20.39 g/bag of the substrate, with a yield of 8.86 under the growing conditions with 50% natural light (100 - 300 lux). Two samples of mushroom alcohol and water extracts were inhibited to negative bacteria strains E. coli, Salmonella spp. and positive strains of S. aureus, B. cereus but did not show inhibition to P. aeruginosa Conflict of interest declaration
The authors declare that there are no conflicts of interest regarding the publication of this paper References
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ULTRASOUND-ASSISTED ENZYMATIC EXTRACTION OF POLYPHENOLS FROM CUSTARD APPLE (Annona squamosa L.) Peel
Duong T. N. Dan, Thanh T. Phan and Huan T. Phan*
Faculty of Chemical Engineering and Food Technology, Nong Lam University, Ho Chi Minh City, Vietnam
*Email: [email protected] Abstract
The objective of the present research work was to valorize the peel of custard apple (Annona squamosa L.) fruit by ultrasound-assisted enzymatic extraction (UAEE) method. The effects of different processing parameters including total UAEE time (60 - 120 min), enzyme concentration (0.15 - 0.6% v/v), and the composition of enzyme mixture (Celluclast 1.5L and Pectinex Ultra SP- L) on the total polyphenol content and antioxidant capacity of the extracts were investigated. The results showed that the antioxidant capacity positively correlated with the total polyphenol content.
The UAEE using enzyme concentration of 0.15% (v/v) with the Celluclast and Pectinex ratio of 2:1 for 90 min resulted in the highest total polyphenol content (29.90 mg GAE/g sample, DW) with DPPH and ABTS antioxidant capacities of 21.77 and 20.91 (mg AAE/g sample, DW), respectively.
Keywords: antioxidants, Custard apple peel, polyphenol, ultrasound-assisted enzymatic extraction.
1. Introduction
Custard apple, scientifically known as Annona squamosa L., is a tropical American species of the family Annonaceae. In tropical markets, custard apple is recognized for its edible fruit which has a green, thin peel layer and a whitish, sweet pulp. Custard apple consists of around 60% fruit pulp, 32% peel, and 8% seed (Chen et al., 2017). The peel of custard apple contains several physiologically active chemicals with antioxidant and antibacterial effects, especially the phenolics (alkaloid, tannin, flavonoid, saponin), which can be extracted by conventional solvent extraction methods (Nguyen et al., 2021; Roy & Sasikals, 2016; Sharma et al., 2013). However, conventional methods are associated with negative environmental impact, high solvent consumption, and longer extraction times (Jovanović et al., 2017).
Recently, various modern extraction methods have been reported for polyphenols extraction, including microwave extraction, ultrasonic extraction, ultrahigh pressure extraction, etc. (Chandraju et al., 2012). Among advanced techniques, ultrasound extraction with sound waves at a low frequency (40 kHz) is commonly used in food technology (Mason et al., 2005). Under ultrasonic effect, solid and liquid particles are vibrated, as a result, solute quickly diffuses from the solid phase into the solvent (Bouhemad et al., 2010). Several probable mechanisms for the ultrasonic enhancement of extraction, such as cell disruption, improved penetration, enhanced swelling, capillary effect, and hydration process, have been also proposed (Chan et al., 2007).
Besides, the breakdown of cell walls is a critical step in the extraction of numerous bioactive compounds found inside cell walls. The use of enzymes such as cellulase and pectinase during extraction improves the hydrolysis of cell wall components as well as the hydrolysis of polysaccharides in the structure to release bioactive compounds, which acts as a foundation for enzyme-assisted extraction (Gardossi et al., 2010). Moreover, the combination of enzymes and ultrasound has been explored for the use in the extraction of natural products from several plants.
An enzyme's ability can be improved by using ultrasonic sound and would not be inactivated by low ultrasonic power. In addition, it makes the enzyme spread evenly (Yachmenev et al., 1998).
Appropriate ultrasonic treatment can create a small change in the molecular conformation of the enzyme, making the enzyme's structure more flexible and reasonable, and resulting in high catalytic activity (Xu et al., 2021). Ultrasonic treatment for a certain time may increase enzyme activity (Vulfson et al., 1991). Enzyme-assisted extraction technique showed faster extraction, higher recovery, and lower solvent consumption as compared to the extraction without enzyme (Entezari
& C. Pétrier, 2005). The present work aimed to recover polyphenolic compounds from the custard apple (Annona squamosa L.) peel by ultrasound-assisted enzymatic extraction (UAEE) method and evaluate the antioxidant capacity of the obtained extract.
2. Materials and methods
Materials: Ripe custard apples (Annona squamosa L.) were obtained from the Tay Ninh province, Vietnam. Raw materials after harvesting were washed and drained. Then, custard apple peel was collected and dried at temperature of 60oC until the moisture content was below 10%. The dried material was ground into a fine powder and sieved through a sieve with a pore size of 0.3 mm. The custard apple peel powder, after preparation, was stored in aluminum bags at -4oC.
Chemicals: Folin - Ciocalteu, 2,2-diphenyl-1-picrylhydrazyl (DPPH reagent), -2,2-Azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS reagent), gallic acid and ascorbic acid were purchased from Merck KGaA (Germany). Water was obtained from a Millipore purification system (USA).
The other chemicals were analytical grade.
Pectinex Ultra SP-L (pectinase with ≥ 3.800 U/ml) and Celluclast 1.5L (cellulases with ≥ 700 U/ml) were from Novozymes (Denmark).
Extraction procedure:
Custard apple peel powder (1 g) was added to distilled water, which was used as the solvent for the extraction, in a 50 mL beaker. The fixed parameters of the experiments with the UAEE were pH 4.5, ultrasonic time of 20 min, ultrasonic temperature of 60oC, ultrasound power of 400W, and solid to solvent ratio (w/v) of 1:15 (w/v). The mixture of Celluclast (C) and Pectinex (P) enzymes was put into an ultrasonic bath with temperature control (Elma, Germany) to be extracted with parameters being investigated as total UAEE time (30 - 120 min), enzyme concentration (0.15 - 0.6% v/v), and composition of enzyme mixture (ratio of 1C:1P, 1C:2P, and 2C:1P). After that, the beaker of the sample was put in a thermostatic tank to deactivate the enzyme by heating at 90oC for 5 min. The mixture of samples was continuously centrifuged (Hettich, Germany) at 5000 rpm at 25oC for 10 min and then filtered through a filter paper (New Star, Φ = 110 mm). Then, the solid was separated and the extract solution was collected for further analysis.
Experimental design:
Single-factor and completely randomized designs are applied in the experiments. Independent variables are UAEE parameters: UAEE time, enzyme concentration, and the composition of enzyme mixture. Meanwhile, TPC, DPPH and ABTS antioxidant capacities were used as response variables. The condition level resulted in the highest level of TPC and antioxidant activity was selected as control variable for the next experiment.
Analysis methods
Total polyphenol content:
The total phenolic content (TPC) of plant extracts was determined using the Folin-Ciocalteu colorimetric method, which is based on an oxidation-reduction process (Singleton et al., 1999;