Abstract-- In this study, drying kinetics, extraction optimization, total phenolic and flavonoid contents, antioxidant activities, and antimicrobial effects of deer apple (Malus trilobata v Eriolobus trilobatus) fruit species were determined. Representing 50% ethanol and 15.86 solvent-sample ratio; total phenolic and flavonoid content, antioxidant activity by DPPH and CUPRAC methods, and inhibition percent were found as 1000.9 mg gallic acid equivalent/kg fresh weight (FW), 469.5 mg catechin equivalent/kg FW, 967.7 mg Trolox equivalent/kg FW, 3.58 mg Trolox equivalent/g FW, 39.85%, respectively. It is shown that the deer apple fruit has antimicrobial activity against Escherichia coli O157:H7, Staphylococcus aureus, Bacillus cereus, and Aspergillus flavus that known as pathogenic or spoilage microorganisms. This study shows that deer apple fruit is a good source of phenolic and flavonoid and has antimicrobial activity. Also, it contributes information about some physicochemical and antimicrobial properties of deer apple fruit and aims to increase the awareness of this fruit species.
Keywords-- Deer apple fruit, extraction optimization, antioxidant activity, antimicrobial properties, drying properties.
I. INTRODUCTION
The deer apple is a small, usually 5-10 meters, and scarce tree species in the Rosaceae family with little recognition in Turkey (Yilmaz and Ok, 2011; Yilmaz and Yuksel, 2014; Korakis et al., 2006). Poiret described deer apple in 1810 as Crataegus trilobata Poiret, and in 1847 by Romoer, it was turned into a monotypic genus by the name of Eriolobus trilobatus (Poir.) (Yilmaz and Ok, 2011; Yilmaz and Yuksel, 2014; Browicz and Karaca, 1993). Today it is used with the name of Malus trilobata (Yilmaz and Yuksel, 2014). Deer apple is seen only naturally in the Mediterranean region in the world. According to the records, this species is found in Greece, Bulgaria, Turkey, Palestine, Lebanon, and Israel (Yilmaz and Ok, 2011; Browicz, 1972; Boratynski et al., 1992). The distribution of deer apple species is between 350-1450 m (Yilmaz and Ok, 2011). In regions where deer apple is growing, summer is long and dry (Yilmaz and Ok, 2011; Yilmaz and Yuksel, 2014). The soil that it grows on it is rocky, limy, or rich in limestone (Yilmaz, 2012). They are usually grown in leafy forests and shrubland as single or clustered (Gultekin et al., 2007).
Deer apple fruits can be used for many reasons such as consuming as fresh and dried fruits, using as herbal tea flavorings, making vinegar and pickle. Deer apple fruit is also used traditionally against various health problems such as cholesterol, shortness of breath, diabetes, and hypertension (Yilmaz and Ok, 2011; Yilmaz and Yuksel, 2014).
Some studies have been done about deer apples, such as the ecology of the species, fruit and seed properties, body form, phenology, and usage by local people. However, a study has not been observed in the food area. Therefore, in this study, we aimed to determine the drying kinetics, extraction optimization, total phenolic and flavonoid contents, antioxidant activities, and antimicrobial effect of this fruit species.
II. METHODS
A. Materials
The fresh deer apple fruits (Malus trilobata v Eriolobus trilobatus) were obtained from the trees in Nuru village (36°25′N 33°35′E) in Silifke district of Mersin province, Turkey. The fruits were at the ripening stage and stained, damaged, and unsuitable fruits were eliminated. The remaining fruits were stored at 4 °C before the analyzes.
B. Drying Profile
Before the drying processes, deer apple fruits were divided into four equal pieces. The drying process was carried out in an oven (Memmert-UF110, Germany) and vacuum oven at 40, 50, and 60 °C. The oven-drying was conducted at 1.3 m/s constant air velocity. The vacuum oven drying was applied at 60 mbar pressure and 2 L/s pump speed using a vacuum pump (EVP 2XZ-2C,
Table 1. Two factors five-level central composite design (CCD) for the optimization of ethanol-water and solvent-sample ratios.
Run |
Ethanol (%) |
SSR |
|
1 |
50.00 |
30.00 |
|
2 |
92.42 |
30.00 |
|
3 |
50.00 |
30.00 |
|
4 |
20.00 |
20.00 |
|
5 |
50.00 |
30.00 |
|
6 |
20.00 |
40.00 |
|
7 |
80.00 |
40.00 |
|
8 |
50.00 |
44.14 |
|
9 |
7.57 |
30.00 |
|
10 |
50.00 |
15.86 |
|
11 |
50.00 |
30.00 |
|
12 |
80.00 |
20.00 |
|
13 |
50.00 |
30.00 |
Zhejiang, China). We weighted the fruits for every 20 minutes until reaching 20% moisture content.
C. Extraction optimization
The seeds were removed, and the sliced deer apples were dried at 55 °C and then ground to powder. We conducted Response Surface Methodology (RSM) with a two-factor-five-level central composite design (CCD) for the optimization of ethanol and solvent-sample ratios (SSR) (Table 1). Data analysis was performed using the Design-Expert software program. Dried deer apples were mixed with pure water at specific ratios and subjected to extractions according to the conditions specified in the experimental design and for 2 hours on a magnetic stirrer at 25°C. At the end of the extraction, the samples were centrifuged at 4200 rpm for 15 min. Supernatants were filtered using Whatman Grade 1 filter paper. The extracts were kept in tightly closed flasks at 4 °C for further analyzes. Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity by DPPH and CUPRAC methods, and antimicrobial activity were analyzed in three parallel and two replicates.
D. Total phenolic and total flavonoid content
The determination of the total phenolic content was based on the method developed by Singleton and Rossi (1965) and modified by Boskou et al. (2006). The extract (0.5 ml) was mixed with 2.5 ml of Folin-Ciocalteu reagent (0.2 N), and after three minutes, 2 ml of sodium carbonate (7.5%) was added. After well mixing, it was incubated at room temperature for 30 minutes in the dark. The absorbance values were measured using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan) at a wavelength of 760 nm. The results were given as mg gallic acid equivalent (GAE) per kg fresh weight (FW) with a linear range of 0.01–0.10 mg/ml (R2=0.993).
Total flavonoid content analyses were performed according to the method used by Zhishen et al. (1999). One ml of the extract was transferred into the test tube, and 4 ml of purified water and 0.3 ml of 5% sodium nitrite (NaNO2) solution were added. After five minutes, 0.3 ml of 10% aluminum chloride (AlCl3) solution was added and mixed, then allowed to stand for 6 minutes. After the addition of 2 ml of sodium hydroxide (1.0 M), the volume was completed to 10 ml with purified water. The absorbance values were measured at a wavelength of 510 nm using a UV-visible spectrophotometer. The results were given as mg catechin equivalent (CAE) per kg of FW with a linear range of 0.01-0.35 mg/ml (R2=0.996).
E. Antioxidant capacity assays
The radical scavenging activity of samples against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was performed according to the method of Singh et al. (2002). The extract (0.1 ml) was transferred to the flasks. For the control group, ethanol was used instead of the fruit sample. Then, 4.9 ml of DPPH solution (0.1 mM) was added to each flask and mixed with the vortex. It was incubated at 27°C for 20 minutes in the dark. The absorbance values were measured with a UV-visible spectrophotometer at a wavelength of 517 nm. The results were given as mg Trolox equivalent (TE) kg of FW with a linear range of 0.05-0.5 mg/ml (R2 =0.996).
The antiradical activity value was calculated as mg TE/kg FW. The inhibition percent was calculated using the following Eq. 1. All analyses were performed at least in triplicate.
. (1)
The copper reducing antioxidant capacity (CUPRAC) of the samples was determined according to the method described by Apak et al. (2004). One ml of copper (II) chloride (CuCl2), 1 ml of neocuproine, 1 ml of ammonium acetate, 0.1 ml of extracted sample, and 1 ml of purified water were added into a flask. The mixture was incubated for 1 hour in the dark. The absorbance values were measured with a UV-visible spectrophotometer at a wavelength of 450 nm. The results were given as mg TE per g of FW with a linear range of 0.05-1.00 mg/ml (R2=0.991).
F. Antimicrobial activity assays
Antibacterial and antifungal effects of 13 extracts prepared for the RSM study were investigated. In the analysis, an ethanol-water mixture was used to prepare all extracts and standard solutions. They were investigated by agar diffusion method against 5 bacteria (S. aureus ATCC 25923, E. coli O157: H7 ATCC 33150, B. cereus FMC19, L. monocytogenes ATCC 19118, S. Typhimurium ATCC 14028) and 3 molds (A. flavus, A. niger, P. carneum). The microorganisms from stock cultures were subjected to 2-step activation treatment at 18 and 24 h (molds at 26-27ºC on PDA solid medium, bacteria at 36-37ºC, Nutrient Broth). 100 μL of each microorganism stock culture was pipetted, sowed by spread plate method, and left for 20 min. Then, 4 equidistant wells (5mm in diameter) were cut (using cork borer) from the agar. Twenty microliters of the samples and negative control (water) solutions were pipetted into the opened wells. The petri dishes were incubated at 27ºC for 5 days for the molds, and at 37ºC for 18-24 h for the bacteria. Then, zones around the wells were measured in mm.
G. Statistical analysis
SPSS v25.0 statistical program was used in the statistical evaluation of the data. The difference and the correlation between groups were determined according to Duncan's multiple range test and Pearson correlation test, respectively. Differences at p<0.05 were considered statistically significant.
III. RESULTS
The deer apples used in this study were selected from the Mersin region. Post-harvest apples are shown in Fig. 1. In this study, drying kinetics, optimization of extraction, total phenolic and flavonoid contents, antioxidant activity by DPPH and CUPRAC methods, and antimicrobial effect were examined using the deer apple samples.
A. Drying profile
The drying process was carried out using two different ovens (normal and vacuum) and three different temperatures (40, 50, and 60 °C). For each temperature value, the drying process was completed at 1840, 1060, and 640 minutes in the oven and 860, 580, and 400 minutes in the vacuum oven, respectively. Drying graphs are given in Fig. 2. It can be seen that the moisture content values in the drying processes decrease closely to linearity with increasing time (R2>0.98). Temperature and method differences for drying at 40, 50, and 60 °C using an oven and vacuum oven were analyzed by comparing slopes with a linear regression model. There is a significant difference between the oven and vacuum oven applications for each temperature value (p<0.001). Drying studies using a vacuum oven reach the target moisture rate in a shorter time than the oven applications. A statistically significant difference was found between each temperature value for the oven and vacuum oven applications separately (p<0.001). The vacuum application increases the vapor pressure gradient between the samples and its surrounding (Kayacan et al., 2018). This helps to decrease drying time. As the temperature value decreases, the drying rate slows down, and it can reach the specified moisture value for a longer time. Similar results were found for the drying of apple slices (Soydan and Doymaz, 2021), kumquat slices (Izli et al., 2018), mushroom slices (Demiray, 2019), and green apple (Doymaz, 2009).
B. Total phenolic-flavonoid content and antioxidant capacity
Before extraction optimization, deer apple seeds were removed and dried at 55 °C and then ground into powder. The resulting powder was extracted according to the two-factor (ethanol-water and solvent-sample) CCD created in the Design-Expert program (Table 1). Extracts were used in total phenolic and total flavonoid contents, DPPH and CUPRAC analyzes, and the results were interpreted with RSM. Figure 3 shows the bioactive component analysis results. The 10th experiment gave the highest results in all bioactive component analyses. Representing 50% ethanol and 15.86 solvent-sample ratios; total phenolic content, total flavonoid content, antioxidant activity by DPPH and CUPRAC method, and inhibition percent were found as 1000.9 mg GAE/kg FW, 469.5 mg
Figure 1. Images of post-harvest deer apples.
Figure 2. Drying graphics of the deer apples in oven and vacuum oven at different temperatures (40, 50, 60 °C).
Figure 3. Bioactive compounds (total phenolic content (TPC) (a), total flavonoid content (TFC) (b), DPPH (c), CUPRAC (d), and inhibition percent (e)) analysis results.
CAE/kg FW, 967.7 mg TE/kg FW, 3.58 mg TE/g FW, and 39.85%, respectively.
Depending on the ethanol percent and solvent-sample ratio, the total amount of phenolic substance in the deer apple was found between 227.6 and 1000.9 mg GAE/kg FW. Li et al. (2014) found that the total phenolic content of wild apples was 5665.8 ± 911.90 mg GAE/kg dry weight. In another study about wild apple fruits (Malus baccata), the total content of phenolic was found to be 1670 mg GAE/kg FW on average (Sharma and Nath, 2016). Aladedunye and Matthäus (2014) studied wild apples and they found the average total phenolic content as 4943 mg GAE/kg dry weight.
In our study, we found the total flavonoid content of the deer apple between 152.2 - 469.5 mg CE/kg FW. Francini and Sebastiani (2013) found that the flavonoid content of apple species was 3032 mg CE/kg shell and 500 mg CE/kg fruit flesh. In another study, the total flavonoid content of wild apples was found as 965.05 mg Rutin Equivalent (RE)/100 g (Li et al., 2014).
In this study, we found the antioxidant activity of deer apple by DPPH and CUPRAC methods between 309.6 – 967.7 mg TE/kg FW, and 1.03 - 3.58 mg TE/g FW, respectively. We found the percent inhibition values of deer apples between 13.6 - 39.9%. Li et al. (2014) determined the antioxidant activity of wild apple species by the DPPH method between 2512.9 - 9705.6 mg TE/kg. Karaman et al. (2013) found that the antioxidant content of apple varieties was 10.94 mg TE/g FW by the CUPRAC method. Ethanol, methanol, and acetone (100%, 70%, and 50% v/v in water) were evaluated as solvents for the extraction of bioactive compounds from fresh and freeze‐dried apple peels in the study. The differences in the results can be explained with differences in the extraction conditions, type of extraction solvent, post-harvesting conditions or storage, age of the trees, and plant varieties.
C. Extraction optimization
Extraction optimization performed using the Design-Expert program, and the analysis results were interpreted first according to the single run and then for the maximum condition of all bioactive compounds. Contour graphs are given in Fig. 4. When the conditions for obtaining the maximum contents of each of the bioactive compounds were analyzed, the optimum ethanol-water ratio was found to be 43.89, 47.74, 48.05, and 45.02 for total phenolic and flavonoid content, antioxidant activity by DPPH and CUPRAC methods, respectively. The optimum solvent-sample ratio was 20.00 for each component. The ethanol ratio was found to be 46.19, and the SSR was found to be 20.00 in the optimization that maximizes all bioactive compounds. There is a strong correlation (r>0.95) between TPC, TFC, antioxidant activity by DPPH, and CUPRAC methods.
D. Antimicrobial and antifungal activity
In this study, we determined the antimicrobial activities of dried deer apples according to different extraction parameters (Table 2 and Table 3). Upon increasing the sol-
Figure 4. Contour graphics for maximum bioactive compounds (TPC (a), TFC (b), DPPH (c) and CUPRAC (d)).
vent-sample and ethanol-water ratios, the inhibition zone also increased. The highest inhibition zone was observed against E. coli, while the lowest inhibition zone was observed against L. monocytogenes. Against B. cereus and S. aureus, at SSR:20 and ethanol ratio:20 no inhibition was observed. In terms of antifungal properties, the inhibition zone was the highest against A. niger, and similarly, it increased with the increasing in the solvent-sample and ethanol-water ratios.
Jelodarian et al. (2013) have reported that some apple varieties are effective on microorganisms of P. aeruginosa, E. coli, K. pneumoniae, and S. epidermidis. In another study, apple peel and pomace extracts using different solvents were used in antimicrobial tests and according to these analyzes, it is stated that they showed an antimicrobial effect against microorganism of P. fluorescens, E. coli DH5α, S. aureus ATCC 10527, L. monocytogenes NCTC 10527, L. lactis DSM 4366 (Agourram et al., 2013). Unnisa et al. (2012) reported that ethanolic apple extracts were effective against S. aureus, P. aeruginosa, E. coli, and K. pneumoniae microorganisms and produced 20.0, 16.0, 15.0, and 10.0 mm inhibition zones, respectively.
Table 3 shows the result of antifungal activity analyses. A. niger was the most sensitive to deer apple extract, while P. carneum was the most resistant mold species considering all the optimization conditions.
IV. CONCLUSIONS
Drying kinetics, extraction optimization, total phenolic and
flavonoid contents, antioxidant activities, and antimicrobial effect of the
deer apple have been determined for the first time in this study. The results
show that deer apple is an excellent source of phenolic and flavonoid. On the
other hand, it has high antioxidant and antimicrobial activity. These results
are important for the contribution to the literature. Further studies can be
made on antimicrobial activity, especially for different microorganisms. In
this study, we aimed to increase the awareness
