ORIGINAL ARTICLE / ORIGINALBEITRAG
https://doi.org/10.1007/s10341-020-00525-7
Comparison of Bioactive Components, Antimicrobial and
Antimutagenic Features of Organically and Conventionally Grown
Almond Hulls
Zehra Tuğba Murathan1 · Armağan Kaya2· Nurcan Erbil3· Mehmet Arslan3· Emel Dıraz4· Şengül Karaman4
Published online: 7 October 2020
© Springer-Verlag GmbH Deutschland, ein Teil von Springer Nature 2020
Abstract
In Turkey, almonds are grown via the following three methods: organic (O), conventional (C) and good agricultural practice (GAP). Almond seeds are mostly consumed as nuts; thus, the researchers have focused different analyses on only seeds. However, in Turkey, unripe green almond hulls are also consumed. Therefore, we studied the contents of some bioactive components, pigments, and malondialdehyde (MDA) and the antimicrobial, antioxidant, and antimutagenic activities of O, C, and GAP ’Ferradual’ (Frd) and ’Ferragnes’ (Frg) almond hull extracts. The highest total ascorbic acid content was found in O Frd (2.1 mg/g fresh weight [FW]) and GAP Frg (2.0 mg/g FW). The total phenolic content and total flavonoid content in all the genotypes ranged from 19.2 and 43.9 mg/g FW and 3.2 and 20.2 mg/g FW, respectively. In general, the antioxidant activity of C Frg and C Frd was low. C Frg had the highest MDA content (8.53 µmol MDA/g FW), whereas O Frg had the lowest MDA content (0.89 µmol MDA/g FW). The samples had varying ratios of chlorophyll a and b. The O samples had more total carotenoid content than the C samples. The antibacterial activity was only observed in the O and GAP Frd extracts. The antifungal activity could not be detected in any of the extracts of samples. Varying antimutagenic activity in Salmonella typhimurium TA 98 strain and content of some phenolics were observed depending on the variety, growing conditions, and dose.
Keywords Almond · Antioxidant · Antimicrobial · Antimutagenic · Phenolic compounds
Vergleich von bioaktiven Komponenten, antimikrobiellen und antimutagenen Eigenschaften in grünen Schalen ökologisch und konventionell angebauter Mandeln
Schlüsselwörter Mandel · Antioxidans · Antimikrobiell · Antimutagen · Phenolische Inhaltsstoffe
Zehra Tu˘gba Murathan ztugbaabaci@hotmail.com
1 Battalgazi Vocational School, Malatya Turgut Özal University, 44210 Malatya, Turkey
2 Faculty of Engineering, Alanya Alaaddin Keykubat University, 07425 Antalya, Turkey
3 Faculty of Health Sciences, Ardahan University, 75000 Ardahan, Turkey
4 Faculty of Arts and Sciences, Kahramanmara¸s Sütçü ˙Imam University, 46100 Kahramanmara¸s, Turkey
Introduction
Almond is a species of the genus Prunus and the subgenus Amygdalus that is commercially grown worldwide (Gomez et al. 2007). It is well adapted to the entire Mediterranean region. In Turkey, it is one of the most valuable crops as it has rich almond (Prunus dulcis L.) genetic resources (Mısırlı and Gülcan2000). In addition, it grows well in dif-ferent regions of Turkey, and its estimated production was 73,230 tons in 2014 (FAO 2017). Each almond genotype has different nutritional values depending on the ecologi-cal conditions such as growing region, cultivation methods, climate, variety, location, and technical and cultural prac-tices (A¸skın et al. 2007). They are an excellent source of lipids, protein, dietary fiber, minerals, tannins, flavonoids,
α-tocopherol, oleic acids, phytosterols, and phenolic acids (Yada et al. 2011; Esfahlan et al. 2010; Xie et al. 2012). The extracts of green almond hull, shell, and seeds have free radical scavenging abilities. The consumption of al-monds protects against diseases such as diabetes, cardio-vascular disease, and cancer (Chen and Blumberg 2008; Jenkins et al.2008).
In Turkey almonds are produced via the following meth-ods: organic (O), conventional (C), and good agricultural practice (GAP). O products are free of pesticide residue and other synthetic substances commonly used in C agriculture, such as inorganic soluble fertilizers (Assis and Romeiro
2002; Aquino and Assis2007). In addition, O products have reduced risks related to chemical contamination (Abadias et al.,2008). O products have better quality and are tastier and healthier than C products (Jensen et al.2012; Araújo et al.2008). GAP seeks to minimize fresh produce contam-ination by recommending science-based “best practices” in areas such as irrigation water quality, manure management, wildlife management, worker health and hygiene, and post-harvest handling (USDA2014).
Almond hulls are used as livestock feed because they are potentially good sources of food (Takeoka and Dao2000). In addition, green almond hulls are consumed by humans in some country. Almond seeds are most widely consumed as nuts; hence, the researchers have focused their analyses on only the seeds thus far. According to the literature reports, several studies have focused on almond hulls (Takeoka and Dao2000; Sang et al.2002; Pinelo et al.2004; Ahmadpour et al. 2011; Meshkini 2016). However, to the best of our knowledge, the differences in the bioactive compounds, an-tioxidant activity, antimicrobial activity, and antimutagenic activity between almond hulls grown in C and O conditions have not been studied. Therefore, this study aimed at de-termining and comparing these properties of O, GAP, and C almond hulls.
Material and Methods
Plant Material
Green fruits of two almond varieties (‘Ferradual’ [Frd] and ’Ferragnes’ [Frg]) produced via O, C, and GAP were col-lected in May 2014 from Adıyaman city, Besni district, located in the southeastern Anatolia region of Turkey. The samples were transferred to the laboratory in polyethylene bags and stored at 4 °C until analysis. The unripe green almond hulls were used in the analysis.
Fruit Weight, Kernel Weight, and Titratable Acidity Ten green fruits and 10 kernel samples were weighed on a digital scale (TX-4202L, Shimadzu, Japan) sensitive to 0.01 g. The acidity rates were determined using the titrimet-ric method according to Cemero˘glu (1992). The obtained titratable acidity was calculated in terms of citric acid as a percentage (%).
Extraction
After the almond hulls were removed, the almond sam-ples (40 g) were homogenized in 200 mL distilled water us-ing an ULTRA-TURRAX disperser (IKA T18, Germany). Subsequently, the mixtures were shaken on a rotary shaker for 72 h at 190 rpm at room temperature. Each extract was centrifuged at 5000 rpm for 10 min, following which the supernatant was collected. Subsequently, the extract was concentrated using a rotary evaporator (SciLogex RE100-Pro, USA). Filter-sterilized and concentrated extracts were frozen and stored at –20 °C until use. The aqueous extracts were used for determining the antimicrobial and antimuta-genic activities.
The methanolic extract was used for the detection of to-tal phenolic content (TPC), toto-tal flavonoid content (TFC), and antioxidant activities. Hull samples (5 g) were homoge-neously mixed with 50 mL of 85% methanol solution and incubated for 24 h, 150 rpm at 30 °C. They were centrifuged at 5000 rpm for 10 min the subsequent day. The supernatant was used for further analysis.
Total Phenolic, Flavonoid, and Ascorbic Acid
Contents
The TPC was determined colorimetrically using the Folin-Ciocalteu method (Singleton and Rossi1965). The TPC of samples was measured using the gallic acid standard. The results are expressed as milligrams of gallic acid equiva-lents per gram of fresh weight (FW). The TFC was deter-mined using a spectrophotometric method (Quettier-Deleu et al. 2000). Rutin was used for calibration and the TFC expressed was as milligram per gram. The determination of the total ascorbic acid content (TAC) was done using a spectrophotometric method (AOAC 1990). The results were expressed as milligram of ascorbic acid content per gram of fruit hull sample.
Antioxidant Activity
The antioxidant activity was detected using the 2,20 -azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods.
2,20-azino-bis(3-ethyl-benzothiazoline-6-sulfonic Acid) Method
The ABTS radical scavenging activity was determined us-ing the method proposed by Re et al. (1999). The ab-sorbance was recorded at 734 nm using a spectrophotometer (Unico S1205 USA) and the percentage of inhibition was calculated. Trolox solutions were used as a reference stan-dard. The antioxidant capacity was expressed as micromole of Trolox equivalents per gram of sample.
Ferric Reducing Antioxidant Power Method
The ferric ions reducing ability was measured according to the method proposed by Benzie and Strain (1996). The absorbance was read at 593 nm using a spectrophotometer. FeSO4solutions were used for calibration. The ferric ions
reducing the ability of per samples was calculated from the linear calibration curve and expressed as µmol Fe II/g equivalents per g of sample.
2,2-diphenyl-1-picrylhydrazyl Method
The DPPH radical scavenging activity was determined us-ing the method proposed by Sanchez-Moreno et al. (1999). The sample was measured at 515 nm using a spectropho-tometer. DPPH radical inhibition by the sample was cal-culated using the following formula: (absorbance control-absorbance sample/control-absorbance control) × 100.
Total Pigment Content
The pigments were extracted from the samples according to the method described by De-Kok and Graham (1989). The absorbance was measured at 470, 645, and 662 nm. Chloro-phyll a (Cha), chloroChloro-phyll b (Chb), and the total carotenoid contents were calculated according to the method described by Lichtenthaler and Wellburn (1983).
Malondialdehyde Analysis
Malondialdehyde (MDA) analysis was performed accord-ing to the method proposed by Heath and Packer (1968). The absorbance of the supernatant was measured at 532
and 600 nm. The MDA amount was calculated by measure-ments made at 600 nm, which were deduced from those at 532 nm; the extinction coefficient was 155 mM–1cm–1.
Identification of Individual Phenolic
Compounds
The phenolic compounds in hull samples were determined by an HPLC analysis (Agilent Technologies 1100 series, Palo Alto, CA, USA). For extraction, 2 g almond hull and 25 mL methanol (containing 1% HCl) was ultrasonicated for 15 min and filtered. Then, it was again ultrasonicated. After the extract was filtered, it was injected into the HPLC system.
The HPLC system is composed of Ecom pomp (Prague, the Czech Republic), Rheodyne injector valve (20 µL), Hewlett-Packard UV variable powerful detector (1100 model) and SGX C18 (5 µL) column (4.6 × 250 mm) (Prague, the Czech Republic). The column temperature was programmed at 25 °C, and the analysis time was 30 min. The conditions for HPLC separation were as fol-lows: 5% formic acid-ddH2O (A) and 100% methanol (B)
initially, raised to 50% A—50% B in 0–15 min, 100% B in 15 min, 100% B in 5 min, 50% A—50% B in 20–30 min. Chlorogenic acid, coumaric acid, naringenin, and naringin were detected at 285 nm; ellagic acid, rutin hydrate, and myricetin were detected at 257 nm; and kaempferol and quercetin were detected at 370 nm. Compounds were iden-tified by comparing their retention time values with those of authentic standards under analysis. Phenolic compounds were expressed as micrograms per gram (µg g–1).
GC-MS analysis was conducted on an Agilent 5975C Mass Spectrometer coupled with an Agilent GC-6890II se-ries. The GC was equipped with HP-88 capillary column (100 m × 250μm× 0.20μm film thickness) and Helium (He).
Antimicrobial Activity
The antimicrobial activity of aqueous extracts of the sam-ples was determined using the agar diffusion method. Eight strains of bacteria (Pseudomonas aeroginosa ATCC 9027, Bacillus subtilis, Enterobacter aerogenes, Bacillus licheniformis, Klebsiella pneumoniae, Staphylococcus au-reus ATCC 6538, Bacillus megaterium DSM 32, and Es-cherichia coli) and three strains of yeast (Yarrowia lipolyt-ica, Candida albicans, and Saccharomyces cerevisiae) were used to evaluate the antimicrobial activity of the almond samples. Agar wells of 11-mm diameter were prepared with a sterilized cork borer; 200μL of extract was added to the wells. A microbial suspension (1%) of each strain hav-ing 106–107colony forming units/mL was added to 15 mL
Table 1 Some pomological features of almond samples
Fruit Weight (g) Kernel Weight (g) Acidity
(%) pH Harvested Time O Frg 8.7 ± 0.1 2 ± 0.02 3.36 ± 0.6 9.9 ± 0.5 12.05.2014 GAP Frg 7.3 ± 0.04 2.2 ± 0.04 3.12 ± 0.03 10.2 ± 0.9 17.05.2014 C Frg 10.2 ± 1.1 3.4 ± 0.6 3.78 ± 0.2 7.6 ± 0.2 19.05.2014 O Frd 9.5 ± 0.9 2.6 ± 0.01 3.45 ± 0.6 8.5 ± 0.01 12.05.2014 GAP Frd 8.1 ± 1.2 3 ± 0.09 3.34 ± 0.9 9.9 ± 0.4 10.05.2014 C Frd 9.9 ± 0.5 3.6 ± 0.02 3.10 ± 0.1 10.5 ± 0.6 10.05.2014
All values are presented as means ± SD (n = 3)
sterile media (Muller-Hinton agar and Sabouraud 2% Glu-cose agar were used for bacteria and yeast, respectively) (Collins et al. 1989; Bradshaw 1992). Erythromycin was used as a positive control and the inhibition zones were measured using a digital ruler.
Antimutagenic Activity
Bacterial Culture and Antimutagenicity Test
Salmonella typhimurium TA 98 and TA 100 strains were used to examine the antimutagenic activity of almond samples according to Ames test with slight modifications. S. typhimurium TA 98 and TA 100 strains were routinely checked to confirm the genetic properties according to Maron and Ames (1983).
Four different concentrations of aqueous extracts (10, 20, 40, and 80μL/plate) were used. Antimutagenic experiments were performed in the absence of S9 mix on S. typhimurium TA 98 and TA 100 strains. 4-Nitro-o-phenylenediamine (4-NPD) and sodium azide (SA) were used as positive controls for TA 98 (10μg/plate) and TA 100 strains (100μg/plate), respectively. After plating the bacterial culture (100μL/ plate), the almond extract and mutagen (4-NPD for TA 98 and SA for TA 100) were added to 2 mL top agar. After gently mixing each plate, minimal glucose agar was poured onto each plate. The plates were incubated at 37 °C for 48–72 h.
Data Analyses
All experiments were performed in triplicate. The results are represented as mean ± standard deviation. All statisti-cal analyses were performed using the SPSS (version 16) software. One-way analysis of variance (ANOVA) and sig-nificant differences between the groups were detected by multiple comparison procedures according to the method proposed by Duncan (1955). The results were considered statistically significant at p < 0.05. Antimutagenic data were
tested for normal distribution (Shapiro-Wilk method) and analyzed with ANOVA and Dunnett’s test.
Result and Discussion
Fruit Weight, Kernel Weight, Acidity, and pH
Some pomological features of almond samples are shown in Table1. All almond samples were harvested from May 10 to May 19 during the green fruit period. The fruit weight ranged from 7.3 and 10.2 g, and kernel weight was be-tween 2 and 3.6 g. The C Frg samples had the highest fruit weight. The acidity and pH values of samples ranged from 3.10 to 3.78% and 7.6 to 10.5, respectively.
Ascorbic Acid, Total Phenolic, Total Flavonoid
Contents and Antioxidant Activities
Pesticides and artificial fertilizer are chemicals that are commonly used in agricultural applications worldwide; these chemicals cause significant health disorders in hu-man beings (Lesueur et al. 2008; Banerjee et al. 2001). In addition, these chemicals are an important stress factor. Pesticide applications increase the reactive oxygen species (ROS) level and damage the plant tissues. The plant has enzymatic and non-enzymatic antioxidants that control the stress factors (Mandal et al. 2009). Ascorbic acid is the most important antioxidant that is effective in scavenging superoxide radical anions, hydroxyl radicals, hydrogen peroxide, reactive nitrogen species, and singlet oxygen (Lykkesfeldt 2000). The European Food Safety Authority recommends a nutrient intake of ascorbic acid between 25 and 45 mg/day, depending on the age (EFSA 2013). Fruits and vegetables are the most important sources of ascorbic acid. In this study, the highest TAC was recorded for O Frd (2.1 mg/g FW) and GAP Frg (2.0 mg/g FW) extracts, whereas the lowest TAC was recorded for C Frd (0.9 mg/g FW) and C Frg (1.1 mg/g FW) extracts (Table2).
Table 2 Ascorbic acid, total phenolic contents and antioxidant activities of almond extracts Total Ascorbic acid Content (mg/g) Total Flavonoid Content (mg/g) Total Phenolic Content (mg/g) DPPH (%) FRAP (µmol Fe II/g FW) ABTS (µmol TE/g FW)
O Frg 1.9 ± 0.01b 20.2 ± 0.002a 43.9 ± 0.001a 80.4 ± 0.46b 77 ± 1a 1.5 ± 0.1a
GAP Frg 2.0 ± 0.03a 5.5 ± 0.01b 30.7 ± 0.007b 89.4 ± 0.92a 74 ± 6.7b 1.3 ± 0.2b
C Frg 1.1 ± 0.08c 3.2 ± 0.01c 21.3 ± 0.002d 57.7 ± 4.58d 70 ± 9.2c 1.3 ± 0b
O Frd 2.1 ± 0.07a 18.2 ± 0.2a 36.7 ± 0.004ab 86.5 ± 1.58a 74 ± 10b 1.4 ± 0.03ab
GAP Frd 1.9 ± 0.03b 4.0 ± 0.03c 24.7 ± 0.003c 54.4 ± 4.65e 75 ± 3b 1.4 ± 0.7ab
C Frd 0.9 ± 0.02d 7.8 ± 0.09b 19.2 ± 0.002e 73.5 ± 9.16c 72 ± 1.6bc 1.3 ± 0.8b
All values are presented as means ± SD (n = 3). Different letters (a–e) within the columns indicate statistically significant differences by Duncan’s multiple range test at p < 0.05
Table 3 MDA and pigment content of almond extracts MDA (µmol/g FW) Cha (µg/g) Chb (µg/g) TC (µg/g) Carotenoid (µg/g) O Frg 0.89 ± 0.17d 3.36 ± 0.13ab 1.69 ± 0.61c 5.05 ± 0.23b 5.95 ± 0.23a
GAP Frg 4.94 ± 0.32b 3.64 ± 0.11a 3.32 ± 0.65b 6.92 ± 0.11a 3.92 ± 0.32b
C Frg 8.53 ± 0.45a 3.00 ± 0.05b 1.29 ± 0.48 cd 4.29 ± 0.22c 3.22 ± 0.33c
O Frd 1.60 ± 0.09c 2.23 ± 0.13d 3.60 ± 0.88a 5.83 ± 0.34ab 3.25 ± 0.45c
GAP Frd 1.77 ± 0.24c 2.53 ± 0.05c 1.89 ± 0.09c 4.42 ± 0.09c 3.17 ± 0.11d
C Frd 2.44 ± 0.17bc 2.78 ± 0.03c 0.27 ± 0.10d 3.05 ± 0.43d 2.83 ± 0.45e
All values are presented as means ± SD (n = 3). Different letters (a–e) within the columns indicate statistically significant differences by Duncan’s multiple range test at p < 0.05
The phenolics and flavonoids are highly effective free radical scavengers and antioxidants. Almond green hull as a potent source of natural antioxidants because it contains large amounts of phenolics and flavonoids (Milbury et al.
2006). Almond polyphenols are mostly found in the hull (Garrido et al. 2008). Almond hulls can limit the risk of various oxidative associated diseases (Meshkini2016). The TPC and TFC of almond hulls are shown in Table2. The TPC in each genotype of almond hull methanol ex-tracts ranged from 19.2 mg/g FW (C Frd) to 43.9 mg/g FW (O Frg), whereas the TFC ranged from 3.2 mg/g FW (C Frg) to 20.2 mg/g FW (O Frg). The TPC and TFC of O Frg hulls were significantly higher than those of other samples. A high pesticide use reduces the phenolic con-tent in plants (Lombardi-Boccia et al. 2004). Previously, researchers have reported that the TPC for almond hulls ranged from 35.9 to 166.7 mg/g dry weight (DW) (Siri-wardhana and Shahidi2002; Wijeratne et al.2006; Sfahlan et al.2009). Meshkini (2016) found that the TPC varied from 62.8 to 74.8 mg/g DW and the TFC varied from 30 to 50.2 mg/g DW in almond hull methanol extracts. Compared to our study, higher values were obtained in other studies. The low value of TPC and TFC in our study can be ex-plained by the use of fresh green hull. On the other hand, it should be considered that the TPC and TFC are influenced by factors such as climate, agricultural conditions, season, and harvesting time.
Some researchers have reported a direct correlation be-tween antioxidant activity, TFC, and TPC (Ferreira et al.
2007). Similarly, the protective effects of natural antioxi-dants in fruits are also related to vitamins and carotenoids. Antioxidants act as scavengers of ROS and free radicals. They decrease the risk of developing different diseases (Thaipong et al.2006). In the present study, the reactivity of almond extracts was analyzed using the DPPH, ABTS, and FRAP assays. Table2shows the antioxidant activities of the extracts. In our study, GAP Frg (89.4%) and O Frd (86.5%) extracts have the highest DPPH activity. Furthermore, O Frg had the highest ABTS activity (1.5 µmol TE (torolox equiv-alent)/g FW) (p < 0.05). FRAP values for the extracts did not statistically differ. In general, the antioxidant activ-ity of C Frg and Frd was low. Pesticide use in C agri-culture possibly reduces the antioxidant activity of hulls. Sfahlan et al. (2009) reported that almonds hull methanol extracts had strong antioxidant activity. According to Pinelo et al. (2004), the percentage inhibition of DPPH radical in almond hull extracts was between 14.92 and 58.05%. A study on almond hull grown in California found a three-fold difference in the FRAP result (210 µmol/g) (Bolling et al.2010). Growing conditions and differences in meth-ods could also affect antioxidant activity of the almonds.
Lipid Peroxidation and Pigment Content
Table3shows the MDA and pigment contents in the almond samples. MDA is the last product of lipid peroxidation and is an oxidative stress indicator. Plant flavonoids inhibit lipid peroxidation. Additionally, the phenolic compounds are in-hibitors of lipid peroxidation (Formica and Regelson1995). Barreira et al. (2008) reported that almond extracts have lipid peroxidation inhibition capacity. In this study, MDA was higher in C samples than in O samples. C Frg had the highest MDA content (8.53 µmol MDA/g FW), whereas O Frg had the lowest MDA content (0.89 µmol MDA/g FW). The consumption of chlorophyll is important in daily diet (Lanfer-Marquez and Barros2005). Hoshina et al. (1998) reported that chlorophylls are important antioxidants that inhibit the lipid autoxidation. Ferruzzi et al. (2002)
con-Table 4 Phenolic composition of almond hulls Phenolic Composition O Frd (µg/g) GAP Frd (µg/g) C Frd (µg/g) O Frg (µg/g) GAP Frg (µg/g) C Frg (µg/g) Chlorogenic acid 140 ± 0.01 29.75 ± 0.11 15.5 ± 0.17 145.25 ± 0.12 235 ± 0.33 236 ± 0.8 Coumaric acid 16 ± 0.03 34 ± 0.3 15.5 ± 0.28 17.5 ± 0.06 16.25 ± 0.01 17.75 ± 0.11 Naringin 22 ± 0.03 116.5 ± 0.4 8.75 ± 0.1 12 ± 0.01 13.25 ± 0.01 9.25 ± 0.03 Naringenin 6 ± 0.01 3.25 ± 0.03 1 ± 0.01 3.75 ± 0.03 6 ± 0.02 13.5 ± 0.02 Routine hydrate 3 ± 0.01 3.5 ± 0.01 5 ± 0.01 2.75 ± 0.02 2.75 ± 0.03 3 ± 0.01 Myrcetin 63 ± 0.25 88.25 ± 0.01 89.75 ± 0.15 45.5 ± 0.04 73 ± 0.03 63.75 ± 0.13 Ellagic acid 34.5 ± 0.12 35.5 ± 0.01 46.5 ± 0.2 21.75 ± 0.5 26 ± 0.33 22.75 ± 0.11 Quercetin 60.25 ± 0.21 – 168.25 ± 0.33 65 ± 0.31 60.75 ± 0.31 70.75 ± 0.01 Campferol 241.25 ± 0.19 231.25 ± 0.44 – 204 ± 0.51 192.75 ± 0.17 211 ± 0.13
All values are presented as means ± SD (n = 3) Table 5 Antimicrobial activities of of almond extracts
Pseudomonas aeroginosa ATCC 9027
Bacillus subtilis Enterobacter aerogenes Bacillus licheni-formis Klebsiella pneu-moniae Staphylococcus aureus ATCC 6538 O Frg NA NA NA NA NA NA GAP Frg NA NA NA NA NA NA C Frg NA NA NA NA NA NA O Frd 12.6 ± 0.3 NA 13.1 ± 0.4 NA 12.9 ± 0.1 NA GAP Frd NA NA NA NA 12.3 ± 0.0 NA C Frd NA NA NA NA NA NA Erythromycin 29.57 ± 0.52 19.19 ± 1.47 25.19 ± 0.34 29.40 ± 0.64 23.16 ± 0.30 22.65 ± 0.46 Bacillus mega-terium DSM 32
Escherichia coli Yarrovia lipolyt-ica Candida albi-cans Saccharomyces cerevisiae O Frg NA NA NA NA NA GAP Frg NA NA NA NA NA C Frg NA NA NA NA NA O Frd 13.6 ± 0.460 NA NA NA NA GAP Frd 12.5 ± 0.323 NA NA NA NA C Frd NA NA NA NA NA Erythromycin 24.41 ± 0.43 22.88 ± 0.43 NA NA NA
All values are presented as means ± SD (n = 3) NA no activity observed
cluded that chlorophylls have antioxidant and antimuta-genic activity. Carotenoids are non-enzymatic antioxidants that play a protective role against oxidative stress. In our study, the total chlorophyll content ranged from 3.05 and 6.92 µg/g FW. The C samples had the lowest total chloro-phyll contents (p < 0.05). The samples had varying ratios of Cha and Chb. The total carotenoid content in O sam-ples was higher than that in C samsam-ples. The highest total carotenoid content of 5.95 µg/g FW was found in O Frg.
Individual Phenolic Compounds
The phenolic profiles of the almond hulls were detected by HPLC (Table 4). Statistically significant differences were observed between the samples (p < 0.05). Table 4
Table 6 Antimutagenicity of almond extracts in Salmonella typhimurium TA 98 strain Extracts Concentration
(µl/Plate)
Revertant colonies Concentration
(µl/Plate) Revertant colonies Mean ± SD Mean ± SD O Frd Control 15.33 ± 2.19 OP Frg Control 15.33 ± 2.19 Positive Control (4-NPD) 1034.0 ± 35.0 Positive Control* (4-NPD) 1034.0 ± 35.0 10 µl/Plate 901 ± 151 10 µl/Plate 655.3 ± 57.0 * 20 µl/Plate 792.7 ± 92.5 20 µl/Plate 663 ± 104 * 40 µl/Plate 594.0 ± 73.5 * 40 µl/Plate 555.3 ± 30.1 * 80 µl/Plate 476.7 ± 36.4 * 80 µl/Plate 536.0 ± 75.7 *
GAP Frd Control 15.33 ± 2.19 GAP Frg Control 15.33 ± 2.19
Positive Control* (4-NPD) 1034.0 ± 35.0 Positive Control* (4-NPD) 1034.0 ± 35.0 10 µl/Plate 510 ± 116 * 10 µl/Plate 642.7 ± 33.0 * 20 µl/Plate 552 ± 104 * 20 µl/Plate 852.7 ± 76.3 40 µl/Plate 488.0 ± 52.5 * 40 µl/Plate 452.0 ± 47.8 * 80 µl/Plate 634.0 ± 17.0 * 80 µl/Plate 698.0 ± 69.5 * C Frd Control 15.33 ± 2.19 C Frg Control 15.33 ± 2.19 Positive Control* (4-NPD) 1034.0 ± 35.0 Positive Control* (4-NPD) 1034.0 ± 35.0 10 µl/Plate 724.0 ± 53.3 10 µl/Plate 607.3 ± 37.8 * 20 µl/Plate 798 ± 219 20 µl/Plate 480.0 ± 96.5 * 40 µl/Plate 696.7 ± 91.0 40 µl/Plate 648.7 ± 50.9 * 80 µl/Plate 707 ± 119 80 µl/Plate 638.7 ± 76.9 *
All values are presented as means ± SD (n = 3) * P≤ 0.05
Table 7 Antimutagenicity of almond extracts in Salmonella typhimurium TA 100 strain Extract Concentration
(µl/Plate)
Revertant colonies Concentration
(µl/Plate) Revertant colonies Mean ± SD Mean ± SD O Frd Control 187.33 ± 6.77 OP Frg Control 187.33 ± 6.77 Positive Control (SA) 2906.7 ± 66.6 Positive Control (SA) 2906.7 ± 66.6 10 µl/Plate 2375 ± 505 10 µl/Plate 3000 ± 205 20 µl/Plate 1927 ± 432 20 µl/Plate 2739 ± 179 40 µl/Plate 1935 ± 169 40 µl/Plate 2843 ± 193 80 µl/Plate 2762 ± 446 80 µl/Plate 3721 ± 412
GAP Frd Control 187.33 ± 6.77 GAP Frg Control 187.33 ± 6.77
Positive Control* (SA) 2906.7 ± 66.6 Positive Control* (SA) 2906.7 ± 66.6 10 µl/Plate 3146 ± 408 10 µl/Plate 2448 ± 460 20 µl/Plate 2937 ± 504 20 µl/Plate 3255 ± 539 40 µl/Plate 2952 ± 301 40 µl/Plate 2525 ± 357 80 µl/Plate 3797 ± 385 80 µl/Plate 2321 ± 418 C Frd Control 187.33 ± 6.77 C Frg Control 187.33 ± 6.77 Positive Control* (SA) 2906.7 ± 66.6 Positive Control* (SA) 2906.7 ± 66.6 10 µl/Plate 2829 ± 497 10 µl/Plate 4499 ± 168 20 µl/Plate 2780 ± 462 20 µl/Plate 2525 ± 611 40 µl/Plate 2063 ± 247 40 µl/Plate 3253 ± 106 80 µl/Plate 2586 ± 788 80 µl/Plate 2877 ± 323
All values are presented as means ± SD (n = 3) * P≤ 0.05
shows that the chlorogenic acid content varied from 0.14 to 236 µg/g, coumaric acid content varied from 15.5 to 34 µg/g, naringin content varied from 8.75 to 116.5 µg/g, naringenin content varied from 1 to 13.5 µg/g, routine hydrate content varied from 2.75 to 5 µg/g, myricetin con-tent varied from 45.5 to 89.75 µg/g, ellagic acid concon-tent varied from 21.75 to 46.5 µg/g, quercetin content varied from 60.25 to 168.25 µg/g, kaempferol content varied from 192.75 to 241.25 µg/g, and catechin content varied from 9.25 to 232 µg/g. The amount of phenolic compounds in fruits is strongly dependent on factors such as the degree of ripeness, variety, climate, soil composition, geographic location, and storage conditions (Belitz et al.2009). Pre-viously, researchers have reported that almond hulls have sinapic acid, caffeic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid (Takeoka and Dao 2003; Siriwardhana et al. 2006). Yildirim et al. (2016) reported that Frd seeds had the highest catechin con-tent (117.59 mg kg–1in 2008 and 145.86 mg kg–1in 2009).
According to Takeoka and Dao (2003), the chlorogenic acid content in almond hulls is 45.52 mg/100 g. Catechin, epicatechin, kaempferol, and isorhamnetin are the major almond skin phenolics and flavonoids (Milbury et al.2006). According to the data obtained, chlorogenic acid and naringenin contents were the highest in C Frg extracts, whereas the lowest value was recorded for O Frd and C Frd extracts, respectively. Coumaric acid and naringin contents were the highest in GAP Frd extracts. The C Frd extracts had the highest routine hydrate, myricetin, ellagic acid, and quercetin contents. Furthermore, the O Frd extracts had the highest kaempferol content, and the GAP Frg extracts had the highest catechin content.
Antimicrobial and Antimutagenic Activity
Agar diffusion method was used to determine the antimi-crobial activities of aqueous extracts. Antimiantimi-crobial activ-ities were tested against 11 different test microorganisms, including 8 bacteria and 3 yeast. The standard antibiotic erythromycin was used as a positive control (Table5). The methanol extracts of Frg samples did not possess antibacte-rial activity against test microorganisms. The antibacteantibacte-rial activity was observed only in the aqueous extracts of O and GAP Frd. O Frd extracts exhibited antibacterial activity against P. aeroginosa ATCC 9027, E. aerogenes, K. pneu-moniae, and B. megaterium DSM 32 with an inhibition zone of 12.62, 13.08, 12.96, and 13.58 mm, respectively. GAP Frd extracts showed antibacterial activity at low rates against only K. pneumoniae and B. megaterium DSM 32 (12.29 and 12.54 mm, respectively). The antifungal activity could not be detected in any of the extracts. Only a few studies are available in the literature regarding the
antimi-crobial activity of the almond. Mandalari et al. (2010) found that almond skin extracts had antibacterial activity against L. monocytogenes and S. aureus (250–500 µg mL–1).
In this study, the antimutagenic activity of almond aque-ous extracts was tested at four different concentrations (10, 20, 40, and 80 µL/plate). The tests were performed in S. ty-phimurium TA 98 and TA 100 strains (Table6and7). All doses of GAP Frd, C Frg and GAP Frg; 40 and 80μL/ plate doses of O Frd; and 10, 40, and 80 µL/plate doses of O Frg exhibited the antimutagenic activity against the S. typhimurium TA 98 strain. None of the concentration of C Frd was found to be statistically significant. None of the almond extracts exhibited antimutagenic effect against the S. typhimurium TA 100 strain. To the best of our knowl-edge, there are only few studies on the antimutagenic ac-tivity of almonds. Yamamoto et al. (1992) reported that almond seed hexan extracts did not have any antimutagenic activity. Zhang et al. (2011) found that the almond seed ex-tracts did not possess antimutagenic against S. typhimurium TA97, TA98, TA100, TA102, and TA1535 strains.
Conclusion
At present, many researchers think that the intake of agri-cultural toxicants may cause health problems; therefore, O foods are more popular than C foods. On the other hand, synthetical chemicals that are used in conventional agricul-tural fields affect the plant development and product quality. The results obtained from this study indicate that the vari-ety and growing system influenced the bioactive compound, MDA and pigment contents and the antioxidant, antimicro-bial, and antimutagenic activities. The TAC, TFC, TPC, total carotenoid, and total chlorophyll contents were found to be higher in O samples than in C samples. The level of phenolic acids varied in all the samples. We could not confirm if a higher phenolic acid content was present in O hulls or C hulls. Almond hull methanolic extracts pos-sess antioxidant activity, especially, O Frd and Frg extracts showed strong antioxidant activities in the DPPH method. C samples generally had low bioactive compounds and pos-sessed low antioxidant activity. Higher MDA content was observed in C Frd and Frg. The aqueous extracts of O and GAP Frd hulls possess antimicrobial properties. The results of this study revealed that pesticide use in C samples re-duces bioactive compounds and antioxidant activity. These findings may be a valuable resource for further physiology, food, agriculture, and medicinal studies.
Acknowledgements We wish to thank the Ardahan University Scien-tific Research Commission for supporting our study through Project grants no. 2015/2.
Conflict of interest Z.T. Murathan, A. Kaya, N. Erbil, M. Arslan, E. Dıraz and ¸S. Karaman declare that they have no competing inter-ests.
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