Impact of Asian Bullfrog (Rana tigerina) Skin Oil on Growth Inhibition of Colletotrichum gloeosporioides (in vitro) and Its Prediction Modeling

Abstract

The effect of Asian Bullfrog (Rana tigerina) skin oil on the mycelium growth of pathogenic fungi, Colletotrichum gloeosporioides, was studied in vitro. A poisoned substrate technique method (dilution in solid media) was employed to evaluate the antifungal activity of the frog skin oil (FSO) at different concentrations (0–10%) against C. gloeosporioides. FSO showed an inhibitory effect, and the efficacy increased with increasing FSO concentration. A modified logistic model was applied to evaluate the antifungal activity of FSO at different concentrations. The modified logistic model showed a good agreement with the observed growth data for C. gloeosporioides. It can be concluded that FSO could potentially control the growth of C. gloeosporioides and that the modified logistic model could describe the inhibitory pattern of FSO against C. gloeosporioides effectively.

Introduction

Many food and agricultural crops encounter deterioration and spoilage caused by filamentous fungi, which are widely dispersed in nature (Nikkhah et al., 2017). During post-harvesting, agricultural crops are susceptible to infection and contamination with some pathogenic fungi (Nikkhah et al., 2017). Contamination by some filamentous fungi is the main cause of the rapid spoilage of fresh fruits, which affects quality and shelf life, especially for fresh fruit (Tejeswini, 2014).

Mango (Mangifera indica L.) is one of the most important tropical fruit crops with significant commercial value (Lawson et al., 2019). Colletotrichum gloeosporioides is a major fungal plant pathogen commonly found in mango fruit that frequently causes massive economic losses during the post-harvesting stage (Klangmuang and Sothornvit, 2018). Therefore, to reduce the risk of massive economic losses, the techniques to control those filamentous fungi and maintain the quality of products during post-harvesting are needed.

Nowadays, safety issues (human health and the environment) caused by the use of chemical fungicides on horticulture products have gained increasing attention. To avoid this issue, the extracted natural products have been used instead of chemical fungicide. (Nikkhah et al., 2017). Several oils have been extracted and studied in regard to their potential to inhibit the growth of various fungi, including peppermint (Mentha piperita L.) oil (Chaemsanit et al., 2018), peppermint essential oil (de Oliveira et al., 2017), and thyme oil (Vilaplana et al., 2018). FSO is known to include some bioactive constituents rich in polyunsaturated fatty acids, such as the omega group. These constituents have been used in many fields, especially pharmaceutical related to medical use (Alencar et al., 2015) and microbiologically relevant for inhibiting food spoilage bacteria (Karnjanapratum et al., 2019a; Karnjanapratum et al., 2019b). Karnjanapratum et al. (2017) reported that the oil extracted from frog (Rana tigerina) skin obtained as a by-product could be an excellent natural alternative antimicrobial agent against both Gram-positive and Gram-negative bacteria which was prepared as emulsion gelatin base film packaging. However, the information of antifungal effect of FSO has not reported.

Predictive microbiology is a useful tool in the food industry (Basak and Guha, 2015). Predictive models can describe the behavior of microorganisms under different conditions, assist process design, and optimize food chain systems from the perspective of microbial safety and extended shelf life (Sosa-Morales et al., 2009). Many models for predicting fungal growth have been developed in the last decade (Basak, 2018; Nikkhah et al., 2017; Serment-Moreno et al., 2017; Basak and Guha, 2015; Tornuk et al., 2013; Dagnas and Membré, 2013; Garcia et al., 2011; Gougouli and Koutsoumanis, 2010). Among these models, modified logistic models are some of the most useful for defining the combination of factors that helps prevent the growth of microorganisms in food systems (Gutarowska and Zakowska, 2009).

To maximize the exploitation of FSO as an alternative natural antifungal agent for application in food and agricultural crops, the efficacy of FSO in inhibiting the growth of mold should be evaluated. Moreover, a quantitative comparison of the effects of FSO concentrations on mold growth rates by mathematical model analysis needs to be implemented for better understanding. Therefore, we carried out a mold growth experiment to quantify the influence of different FSO concentrations on the mycelial growth of mold (C. gloeosporioides) incubated on PDA medium, and employed a modified logistic model to evaluate the growth curve of C. gloeosporioides at various FSO concentrations.

Materials and Methods

(1) Chemicals    All chemicals were of analytical grade. Potato dextrose agar was obtained from Merck (Darmstadt, Germany). Tween 20 was purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA).

(2) Frog skin preparation    Asian bullfrogs (R. tigerina; weight: 200–300 g) were obtained from a local market in Ladkrabang, Bangkok, Thailand. The frog skins were removed and washed with iced tap water (1–3 °C), and then pooled as a composite sample in a polyethylene bag and stored at −20 °C until use, in accordance with a previous study (Karnjanapratum et al., 2019b). Prior to FSO extraction, the frozen skins were cut into small pieces (about 1 × 1 cm²) using scissors and pulverized in a blender in the presence of liquid nitrogen (Phillips, Guangzhou, China).

(3) Extraction of FSO    FSO was extracted as follows in accordance with a previous study (Bligh and Dyer, 1959). Samples (100 g) were mixed and homogenized with 800 mL of a mixture of chloroform:methanol:water (1:2:1, v/v/v) at 11 000 rpm for 1 min. Then, 200 mL of chloroform was added, and the mixture was homogenized for 1 min. Thereafter, 100 mL of water was added to the mixture and homogenized for 30 s at the same speed. Next, the mixture was filtered through Whatman No. 4 filter paper (Whatman International Ltd., Maidstone, UK). The filtrate was transferred into a separating funnel where the chloroform phase (bottom phase) was drained off into an Erlenmeyer flask. Sodium sulphate (anhydrous) (10–12 g) was added, and the mixture was shaken thoroughly to remove the residual water. Lipid in chloroform was decanted into a round-bottomed flask through filter paper (Whatman No. 4). The chloroform was then evaporated at 45 °C using a rotary evaporator (Rotavapor R-14; Büchi, Tokyo, Japan). The residual solvent was removed by flushing with nitrogen. The lipid was transferred to an amber vial and the sample was kept at −20 °C until analysis.

(4) Culture mold    The mold (C. gloeosporioides) was purchased from the Thailand Network on Culture Collection, DOAC Culture Collection Centre, Plant Pathology and Microbiology Division, Department of Agriculture, Thailand, and cultured on PDA for 7 d at 25 ± 2 °C. Stock cultures were maintained on PDA slants at 25 ± 2 °C for further study. The fungi were sub-cultured on fresh PDA and incubated at 25 ± 2 °C for 10 d before the experiments. To prevent the growth of bacteria, 10% tartaric acid (Sigma-Aldrich, Steinheim, Germany) was added to the fungi culture media.

(5) In vitro experiment    The effects of FSO on the radial mycelial growth of the tested (C. gloeosporioides) strains were assessed using the poisoned substrate technique (dilution in solid media) (de Oliveira et al., 2017). The PDA solution was sterilized by autoclaving at 121 °C for 15 min. Then, FSO was added to the warm PDA at the desired final concentrations (0%, 2%, 4%, 6%, 8%, and 10%) containing 5% of Tween 20 (v/v), and then poured into glass Petri dishes (90 mm in diameter) at a temperature of 40–45 °C. PDA containing sterilized water was similarly tested as a control. Next, one agar plug (5 mm in diameter) containing C. gloeosporioides mycelia, which was taken from the margin of a 10-d old culture, was transferred to the center of each plate using a cork borer. Each treatment was performed in triplicate. Using a vernier caliper, radial mycelial growth was measured every 2 d intervals until 8 d. or until the control Petri dish was fully covered with mycelia. The radial mycelial growth diameter (mm) of the strain colony was measured in two perpendicular directions, and the colony mean diameter was obtained.

(6) Modeling of fungal growth

a) Modified logistic model    The modified logistic model is sigmoidal, which is more appropriate for fitting growth curves complete with all three phases: lag, exponential, and stationary (Tornuk et al., 2013). The modified logistic equation was adopted from Koide and Yasokawa (2008) to describe the nonlinear inactivation of microorganisms:   

where N(t) is the average colony diameter (mm) at time t (d), A is the maximum growth achieved during the stationary phase, µ is the maximum specific growth rate (1/d), and λ is the lag phase (d).

b) Model validation    The model parameters were estimated by regression analysis using Microsoft Excel software (Excel 2016, Microsoft, Redmond, USA). The root-mean-square error (RMSE) was used to evaluate the model. The parameters provide information to identify the differences between the experimental data and the model estimates (Ozcakmak and Gul, 2017). The RMSE was calculated as follows:   

where the value of the experimental data is given by N_exp, the value estimated by the model is given by N_cal, and n is the number of experimental observations.

(7) Statistical analysis    Analysis of variance was used to evaluate the effects of FSO during the incubation period. When a significant (P < 0.05) effect was found, the mean values were further analyzed according to Duncan's multiple range test.

Results and Discussion

(1) In vitro antifungal activity of FSO    The antifungal activities of FSO on the growth of the major fungal pathogen, C. gloeosporioides, at various levels were measured using the direct contact method. The changes in the average colony diameters are shown in Figure 1 and Table 1. The obtained results showed that the growth of C. gloeosporioides depended on the FSO concentrations, with significant differences (P < 0.05). After increasing the incubation time, the highest mold growth rate was found in the control sample, whereas the growth rate decreased in FSO concentrations in the order of 2%, 4%, 6%, 8%, and 10%. FSO droplets could disperse in the PDA matrix. FSO might be binding with vital nutrients for mold growth, thereby retarding the mold growth. These results are in agreement with a previous report on betel leaf (Piper betle L.) essential oil (Basak and Guha, 2017). Moreover, FSO might contain antimicrobial constituents that help prevent the growth of mold. FSO extracted from adipose tissue of frog skin, which is well known as a source of saturated and polyunsaturated fatty acids related to antifungal properties. The mechanisms underlying the inhibition of mold growth are strongly related to the lipophilic characteristics of major components of FSO. Lipophilicity refers to the ability of a chemical component to dissolve in fats, oils, lipids, and non-polar solvents. Some components possess a lipophilic molecular structure, which enables them to penetrate both cellular and mitochondrial membranes, which leads to membrane disruption, cytoplasmic leakage, cell lysis, and cell death (Karnjanapratum et al., 2019b). The potential of FSO in terms of antimicrobial properties has also been documented by some researchers. FSO in the concentration range of 250–500 mg/mL has shown inhibitory activities on Gram-positive Bacillus cereus and Gram-negative Escherichia coli bacteria (Karnjanapratum et al., 2019a; Karnjanapratum et al., 2019b). Silva (2018) also showed that FSO could inhibit the growth of different Candida spp. These results indicate that FSO may be a new natural antifungal agent that can be used as an alternative in food or agricultural products.

Fig. 1.

Mycelium of Colletotrichum gloeosporioides grown on PDA supplemented with FSO at concentrations of 0%, 2%, 4%, 6%, 8%, and 10% during storage at 25 °C for 2, 4, 6, and 8 d.

Table 1. The average diameter with standard deviation (±) of C. gloeosporioides grown on PDA supplemented with FSO at concentrations of 0%, 2%, 4%, 6%, 8%, and 10% during storage at 25 °C for 2, 4, 6, and 8 d.

Days	Diameter (mm) at various FSO concentrations (%)
	Control (0%)	2	4	6	8	10
2	19.26±0.56aD	11.89±0.72bD	11.91±0.46bD	11.50±0.55bD	10.49±0.89bD	7.17±0.30cD
4	28.47±0.43aC	21.39±0.15bC	20.69±0.27bC	18.97±0.08cC	18.27±0.15cC	17.09±0.74dC
6	55.43±1.42aB	41.34±0.82bB	40.05±0.03bcB	38.80±0.40cdB	37.96±0.22deB	36.82±0.25eB
8	70.16±0.14aA	56.54±1.00bA	53.96±0.35cA	51.88±0.49dA	49.94±0.36eA	47.89±0.23fA

Different upper and lower case letters indicate significant difference (p < 0.05) for column and row, respectively.

(2) Mathematical model for fungal growth    To quantify the antifungal effects of FSO, a modified logistic model was employed for predicting the changes in the mycelial diameters of C. gloeosporioides exposed to different FSO concentrations. Figure 2 shows the fitted results of the employed model. The estimated model parameters and RMSEs are listed in Table 2.

Fig. 2.

Fitting of modified logistic model to the experimental data obtained from the mycelium diameter of Colletotrichum gloeosporioides growing on PDA mixed with frog skin oil at various concentrations during storage at 25 °C for 2, 4, 6 and 8 d. The line corresponds to the estimated values from the models distribution function which frog oil concentrations represented by (—) 0%, (—·—) 2%, (---) 4%, (⋯··) 6%, (— —) 8%, (—··—) 10%. The marks correspond value from the experiment which frog oil concentrations represented by (x) 0%, (●) 2%, (○) 4%, (▴) 6%, (△) 8%, (◆) 10%. Error bars represent the standard deviation of 3 experimental values.

Table 2. Parameters and root value of mean squared error (RMSE) obtained from modified logistic model for the growth curve of C. gloeosporioides.

Model	Concentration (%)	Parameter
		A	µ	λ	RMSE
Modified logistic model	0	87.02	10.94	1.1	3.48
	2	71.92	9.39	1.66	1.78
	4	68.14	8.87	1.56	1.74
	6	65.46	8.68	1.65	1.96
	8	60.69	8.62	1.71	1.82
	10	53.87	9.45	2.13	1.06

The modified logistic model has been widely used for predicting mold growth curves (Gutarowska and Zakowska, 2009; Marín et al., 2008). As is well known, a model with sufficient parameters can offer adequate mathematical and biological significance (Chen and Zhu, 2011). The growth pattern obtained from the observed and estimated data followed a sigmoid curve (lag, log or exponential, diminishing, and stationary phases) (Fig. 2) with satisfactory RMSE. A good fit of the growth curve with the satisfied statistic is important to obtain valid estimates of the modeled parameter (Torres-Ossandón et al., 2019). The maximum growth achieved parameter (A) decreased with increasing FSO concentrations. This parameter is employed as an index of microbial development. A decrease in the maximum growth rate (µ ) and an increase in the apparent lag time (λ) were observed with increasing FSO concentrations (Table 2). These results agreed well with those reported by Basak and Guha (2015) and Basak and Guha (2017), who reported that a decrease in the maximum growth rate (µ ) and an increase in the apparent lag time (λ) were observed with increasing concentrations of betel leaf essential oil. At an FSO concentration of 10%, the maximum growth rate (µ ) was negligibly higher than that at other concentrations. This negligible difference might be caused by deficiencies in the data. Further investigations are needed to clarify this issue. However, the results of the present study suggest that a modified logistic model is an adequate mathematical model to quantify the maximum growth achieved, growth rate, and apparent lag time of C. gloeosporioides under the influence of FSO.

Conclusion

FSO extracted from Asian bullfrogs (R. tigerina) could become an alternative natural antifungal agent possessing inhibitory activity against C. gloeosporioides. The antifungal activity of FSO against C. gloeosporioides increased with increasing concentrations. To quantify the growth behavior, a modified logistic model was employed, and the model parameters A, µ , and λ were determined according to the fitting method. As a result, the modified logistic model was useful for describing and quantifying the behavior of the mold growth on PDA supplemented with various FSO concentrations. The results also indicated that the growth of C. gloeosporioides decreased with increasing FSO concentrations. A decrease in the maximum growth rate (µ ) and an increase in the apparent lag time (λ) were observed with increasing FSO concentrations, with a few exceptions. Since FSO obtained as a byproduct was found to be a potent antifungal agent, it can be expected to be used as a natural preservative coating material to extend the shelf life of food products.

Acknowledgements    The work was supported by the JICA Innovative Asia scholarship [Project no. D1706943] and Thailand Research Fund and Office of the Higher Education Commission [Grant number MRG 6280115].

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