Effects of Thymus vulgaris Essential Oil on Decay Resistance and Quality of Iranian Table Grape
Mahsa GERANSAYEH
1, Younes MOSTOFI
2, Vahid ABDOSSI
11Azad University of Tehran, Science and Research Branch, Tehran, Iran; [email protected] (*corresponding author)
2University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
Abstract
Because of greater consumer awareness and concern regarding synthetic chemical additives, foods preserved with natural additives have become popular. Medicinal plants have been used by human being since ages in traditional medicine due to their therapeutic potential and the search on medicinal plants have led the discovery of novel drug candidates used against diverse diseases. Therefore Thymus vulgaris essential oil was applied in ‘Bidaneh Qermez’ grape cultivar at six concentrations (0, 100, 200, 300, 400 and 500 µl/l) in water. Quality characteristics (pH, decay, vitamin C, reducing sugars, weight loss, berry abscission, dehydration of rachis, berry cracking and sensory analyses) were evaluated. The results showed that treated fruits with essential oil had lower decay percentage, dehydration of rachis, berry abscission, berry cracking and higher pH, reducing sugars and storage quality compared to control. As a general result, essential oil treatment caused lower decay incident and longer storability.
Keywords: essential oil, Lamiaceae, perishable fruit, postharvest attributes, shelf-life
Introduction
Because of greater consumer awareness and concern regarding synthetic chemical additives, foods preserved with natural additives have become popular. This has led researchers and food processors to look for natural food additives with a broad spectrum of antimicrobial activ- ity (Marino et al., 1999). Recently there has been con- siderable interest in generally recognized as safe (GRAS) compounds. Plant essential oils are an example of GRAS compounds (Jobling, 2000). Essential oils are extracted from various aromatic plants. They are liquid, volatile, limpid, and rarely colored, lipid soluble and soluble in or- ganic solvents with a generally lower density than that of water (Bakkali et al., 2008). Some plant essential oils have been reported to have antimicrobial activity against a wide range of spoilage and pathogenic bacteria (Kivang et al., 1991) and often have, in common, active phenolic groups (Holley and Patel, 2005). The efficacy of essential oil from clove and cinnamon against 6 fungi causing postharvest decay of grapes were studied. The best concentration to inhibit growth of 6 fungi was 400 mg/ml (Sukata et al., 2008). A total of 14 odoriferous angiospermic essential oils were tested against the toxigenic strain of Aspergillus flavus and Botrytis cinerea. The essential oil of Thymus vul- garis L. showed highest antifungal efficacy (Kumar et al., 2008). Microbial spoilage counts and losses of quality in term of sensory, nutritional and functional properties were significantly reduced in packages with added eugenol or
thymol in table grapes (Valero et al., 2006). In this study the essential oil of Thymus vulgaris L. has been investigated regarding its potential to inhibit pathogens production and safety for humans. In addition, the effects of Thymus vulgaris on quality-related attributes of table grapes were evaluated.
Materials and methods Plant material
Table grapes (Vitis vinifera L.), cv. ‘Bidaneh Qermez’
were harvested randomly from a commercial vineyard lo- cated in Takestan, Qazvin, Iran at commercial maturity and transported to the laboratory. Grape clusters were divided into small clusters of approximately 300 g each, those with defects were discarded.
Essential oil treatment
Treatments with Thymus vulgaris essential oil (pur- chased from Zardband company, Tehran, Iran), were per- formed by dissolving the requisite amounts of Thymus vulgaris (0, 100, 200, 300, 400 and 500 µl/l) in 25 ml of 0.05 % tween-80 and then mixing with 475 ml of water.
Grapes were dipped in the solutions for 1 min at room temperature and air dried (Hadizadeh et al., 2009). Each treatment was replicated three times with 300 g fruits per replicate. All packages were stored at 4°C and 75 % RH in darkness for 75 days. Measurements were made at room temperature every 15 days.
Received 21 June 2012; accepted 30 September 2012
added three drops of methylene blue indicator (Saini et al., 2006).
Decay percentage
Percent of decay was scored on a 1-9 scale, where: 0=
intact fruit, 1= less than 10% Decay, 2= between 10-20%
decay, 3= between 20-30% decay, 4= between 30-40% de- cay, 5= between 40-50% decay, 6=between 50-65% decay, 7= between 65-80% decay, 8= more than 80% decay (Ni- gro et al., 2000).
Berry abscission, dehydration of rachis and berry cracking
Berries abscission was scored on a 1-5 scale, where: 1=
very low, 2= low, 3= moderate, 4= high, 5= very high.
For the rachis, symptoms of dehydration and browning for primary and secondary branches were evaluated on a ranked scale of 1-5, where= 1= green and fresh, 2= green, 3= semi-dry, 4= 50% dry, 5= absolutely dry (Valero et al., 2006; Xu et al., 2007). Number of cracked berries in 300 g was evaluated (Xu et al., 2007).
Sensory evaluation
Sensory analyses to compare the quality of treated and control table grapes were carried out by a 10 trained adults aged 25-40 years. It was about aroma, taste, firmness, ap- pearance and texture. Panelists scored grapes between 1-10. 10 being the best total quality and 1 being the worst (Martinez-Romero et al., 2007). Assessments were contin- ued until fruits condition were considered unacceptable.
Statistical analysis
Statistical analysis of the data obtained in the present study was carried out using split factorial method in a com- pletely randomized design layout with 3 replications. Data obtained were subjected to analysis of variance (ANOVA).
Means were separated by SNK and LSD test.
Results and discussion
Chemical composition of the essential oil was reported in Tab. 1, with their retention indices (RI), and the per- centages of compounds.
pH rate
While time passed, in storage period, pH rate of grape juice increased (Tab. 2). Results showed that a significant increase in pH rate was experienced in samples which were treated with essential oil compared to controls (Fig. 1).
pH of the grape extract is determined by changes in the amount of tartaric acid, it is expected that a relationship exists between the level of acidity and pH. When the level of acidity cuts down, pH of fruits extract increase. pH lev- els of strawberries and tomatoes with eucalyptus and cin- namon essential oils didn’t change during storage period (Tzortzakis, 2007).
Gas Chromatography-Mass Spectrometry analysis The analysis of the volatile constituents were run on a Heweltt-Packard GC/MS system (GC: 6890; MS: 5973).
The fused- silica hp INNOWAX capillary column (30 m
× 0.25 mm ID, film tickness of 0.32 µm) was directly cou- pled to the MS. The carrier gas was helium, with a flow rate of 1 mm/min. Oven temperature was programmed (60°C for 3 min, then 60-220°C at 5°C /min) and subsequently, held isothermal for 2 min. Injector temperature: 250°C, detector temperature: 300°C. Split ratio 1:20. Volume in- jected: 0.1 µl of 1% solution (diluted in hexane). The mass spectrometer was hp recording at 70 eV; scan time 1.5 sec;
mass range 40-300 amu.
Identification of components
The components of the oil were identified by compari- son of their mass spectra with those of a computer library (Wiley 275 library). Retention indices were calculated us- ing retention times of n-alkanes that have been injected to the same instrument (Adams, 1995; Shibamoto, 1987).
pH rate
pH was measured using a pH meter Metrohm Lab 827 (Saini et al., 2006).
Weight loss percentage
The effect of essential oil exposure on table grape weight loss was also investigated. Weight loss of individual bunches was recorded on the day of harvesting and after the different sampling dates expressed as percentage loss of original weight (Valero et al., 2006).
Color changes
Color changes of berries were analyzed on the skin surface with a Minolta Chromameter CR400. Color was measured at the further two points apart on the equa- tor of each berry and expressed as Chroma (Chroma = [(a2+b2)0.5]) and Hue angle (H° = tan -1 (b*/a*) (Valero et al., 2006).
Vitamin C content
The content of vitamin C was determined using in- dophenol procedure. 10 ml of samples were filtrated and titrated against sodium 2, 6-dichlorophenol indophenol dye to a faint pink color which persisted for 5-10 seconds.
It was expressed as mg vitamin C/100g fruit weight (Titer x dye equiv. x dilution x100/ Wt. of sample) (Saini et al., 2006).
Reducing sugars content
Reducing sugar was measured with the procedure Lane-Eynon. Mixed Fehling’s solution was made by trans- ferring 5 ml of solution A and 5 ml of solution B to a 250 ml conical flask and mixed well. Diluted fruit juice was added to mix Fehling’s solution drop by drop. It was heat- ed over Bunsen burner and boiled for two minutes. Then
Decay percentage
During the storage period, the amount of decay in- creased, so that in the 75th day the rate of decay reached 7.33 % (Tab. 2). The controls with the mean of 4.28 % had the highest percent of decay. Increasing the concentration
of essential oil resulted in an increase in decay resistance of samples (Fig. 2). The samples which were treated with concentration 500 µl/l endured more than other groups, up to the 75days. These samples did not show any sign of decay up to the 75 days. The use of 1 µl/ml Thymus vulgaris essential oil could significantly enhanced plant resistance to Botrytis cinerea (Kumar et al., 2008). Higher concentra- tions of Thymus vulgaris essential oil led to greatest control of bacteria (Celikel and Kavas, 2008). Lemongrass essen- tial oil could significantly control B. cinerea. Higher con- centration of essential oil led to the greatest decay control (Tzortzakis and Economakis, 2007). Essential oils antimi- crobial activity related to their hydrophobic property and they have high antioxidant role within the plants
(
Brul and Coote, 1999).Vitamin C
Vitamin C content of grapes decreased significantly along the storage (Tab. 2). The samples which were treated with 300 and 400 µl/l essential oil had the lowest vitamin
C compared to others (Fig. 3). No difference was observed between the control and essential oil treated samples with concentration 100, 200 and 500 µl/l in regard to vitamin C content. Vitamin C content of treated fruits with eugenol and thymol decreased along the storage. In treated samples Tab. 2. The mean of evaluated parameters during the storage period in table grape cv. ‘Bidaneh Qermez’
(Day)Time pH Decay (%)
Vit. C (mg/100 g fruit weight)
Reducing
sugars (%) Weight loss (%) Hue
angle Chroma Berry
abscission Rachis
dehydration Berry
cracking (g) Fruit quality
0 3.55d 0.0e 15a 13.92e 0.00e 46.1a 9.77a 0.00e 1.00e 0.0c 10a
15 3.63c 0.5d 13b 14.33d 0.10d 41.4b 9.21b 2.11d 2.50d 0.0c 7.53b
30 3.74bc 1.7c 10c 15.02c 0.14c 40.2bc 7.97c 2.89cd 3.22c 334b 7.50b
45 3.79b 1.7c 10c 15.02c 0.14c 40.5bc 7.02d 3.27c 4.33b 432a
60 3.85a 4.0b 9.6cd 15.71b 0.21b 39.0c 6.74de 4.00b 4.89a 430a
75 3.91a 7.3a 8d 17.04a 0.23a 38.9c 6.50e 4.67a 4.98a 437a
Tab. 1. Chemical composition of the T. vulgaris essential oil Chemical
composition Percentages of
compounds Retention
time Retention indices
Alpha-pinene 1.53 10.45 939
Camphene 0.21 10.90 954
Beta-pinene 5.24 11.63 979
Myrcene 0.98 11.73 991
Alpha-phellandren 0.2 12.25 1003
Alpha-Terpinene 1.34 12.54 1017
Para cymen 19.79 12.80 1025
Limonene 0.92 12.88 1029
Beta-phellandrene 0.53 12.94 1030
Gamma-Terpinene 20.28 13.64 1060
Terpinolene 0.26 15.36 1076
Para-menthatriene 0.1 15.48 1110
Estragole 1.23 16.80 1179
Bornyl formate 0.13 17.62 1289
Thymol 48.28 18.50 1290
Carvacrol 2.54 18.92 1299
Hexa decanoic 0.43 28.11 1600
Fig. 1. The effect of essential oil treatment on pH of ‘Bidaneh
Qermez’ table grape Fig. 2. The effect of essential oil treatment on decay percentage of ‘Bidaneh Qermez’ table grape
Weight loss percentage
Weight loss percentage of fruits increased in the stor- age period gradually (Tab. 2). There was no significant dif- ference among different concentrations of essential oil and controls in regard to weight loss except samples treated with 200 and 400 µl/lessential oil (Fig. 5). It seems that es- sential oil did not affect weight loss of samples. The rate of weight loss of treated tomatoes and strawberries with es- sential oil and control was low and there was no significant difference between them (Tzortzakis, 2007). Weight loss of fruit occurs due to water loss and reduction in stored material via respiration.
Hue angle
The hue angle decreased along the storage (Tab. 2). The samples which were treated with 100 and 200 µl/lessen- tial oil and controls had the highest hue angle compared to others (Fig. 6). Essential oil did not affect significantly color of treated strawberries and tomatoes (Tzortzakis, 2007). Treatments that reduce the rate of fruit ripening caused hue angle reduction (Thumula, 2006).
more vitamin C was reduced compare to controls (Valero et al., 2006). L-ascorbic acid (AA) is the principal bio- logically active form of vitamin C. Fruits show a gradual decrease in AA content as the storage duration increases (Adisa, 1986).
Reducing sugars content
While time passed, reducing sugars rate increased (Tab. 2). Results showed that increase in the concentra- tion of essential oil, led to an increase in reducing sugars content (Fig. 4). Samples with 500 µl/lessential oil had the highest reducing sugars content compared to others.
Reducing sugars in grapes which were treated with thymol and eugenol decreased significantly compared with con- trols. This finding was inconsistent with results obtained in experiment. Reducing sugars of kiwifruits increased gradually at first 7 days then decreased (Wang and Buta, 2003). When reducing sugars decrease, gray mold percent of grapes increase (Hill et al., 1981). It can be concluded that the increase in reducing sugars can enhance plant re- sistance against pathogens, because it can affect antioxi- dant systems of fruit (Rolland et al., 2006).
Fig. 3. The effect of essential oil treatment on vitamin C of ‘Bi- daneh Qermez’ table grape
Fig. 4. The effect of essential oil treatment on reducing sugars of
‘Bidaneh Qermez’ table grape
Fig. 5. The effect of essential oil treatment on weight loss of ‘Bi- daneh Qermez’ table grape
Fig. 6. The effect of essential oil treatment on hue angle of ‘Bi- daneh Qermez’ table grape
concentration of essential oil may prevented production of ABA and resulted in lower berry abscission. Besides, reduction of respiration rate and water loss resulted in re- duction in berry abscission in treated fruits.
Dehydration of rachis
Result showed that while time passed dehydration of rachis increased to 4.98 at the end of the storage period (Tab. 2). Treated samples with 500 µl/l concentration preserved their freshness of the rachis longer than other samples (Fig. 9). It appears that as time passed and as the rate of berry and rachis respiration increased, more water was removed from their tissues and dehydration of rachis appeared.
Berry cracking
During the storage period, the amount of cracked ber- ries increased (Tab. 2). Treated samples with 500 µl/l con- centration had the lowest berry cracking and there was no significant difference among control and treated samples Chroma
The chroma reduced along the storage (Tab. 2). There was no significant difference among samples treated with 100 and 200 µl/lessential oil and controls (Fig. 7). Only the samples with concentration 500 µl/lshowed a sig- nificant decreased in chroma rate with the mean of 7.81.
When the samples experience weight loss and dehydration during the storage period, the phenomenon of browning berries occurs and cause more color changes in them. Re- duction in the amount of green pigment over time is the main cause of chroma reduction (Artes-Hernandez and Aguayo, 2004).
Berry abscission
Results showed that there was a significant increase in berry abscission along the storage (Tab. 2). The maxi- mum berry abscission was observed in controls, whilst the samples with concentrations 400 and 500 µl/lshowed the lowest berry abscission (Fig. 8). ABA content in the pedi- cle with out berry was much higher than the pedicle with berry attached (You-Mei et al., 2000). It seems that high Fig. 7. The effect of essential oil treatment on chroma of ‘Bi- daneh Qermez’ table grape
Fig. 8. The effect of essential oil treatment on berry abscission of
‘Bidaneh Qermez’ table grape
Fig. 9. The effect of essential oil treatment on dehydration of ra- chis of ‘Bidaneh Qermez’ table grape
Fig. 10. The effect of essential oil treatment on berry cracking of
‘Bidaneh Qermez’ table grape
Artes-Hernandez F, Aguayo E (2004). Alternative atmosphere treatments for keeping quality of autumn seedless table grapes during long term cold storage. Postharvest Biol Tech- nol 31:59-67.
Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008). Bio- logical effects of essential oils-A review. Food Chem Toxicol 49:446-457.
Brul S, Coote P (1999). Preservative agents in foods made of action and microbial resistance mechanisms. Internat J Food Microbiol 50:1-17.
Celikel N, Kavas G (2008). Antimicrobial properties of some es- sential oils against some pathogenic microorganisms. Czech J Food Sci 26:174-181.
Considine JA, Kriedemann PE (1972). Fruit splitting in grapes:
determination of the critical turgor pressure. Austral J Agr Res 23:17-24.
Hadizadeh I, Peivastegan B, Hamzeh Zarghani H (2009). An- tifungal activity of essential oils from some medicinal plants of Iran against Alternaria alternate. Am J Appl Sci 6:857- 861.
Hill GS, Kitleer F, Huth G, Schlosser E (1981). Resistance of grapes in different development stages of Botrytis cinerea.
Phyto Pathol Zeitch 102:328-338.
Holley RA, Patel D (2005). Improvement in shelf life and safely of perishable foods by plant essential oils and smoke antimi- crobials. Food Microbiol 22:273-292.
Jobling J (2000). Essential oils: A new idea for postharvest dis- ease control. Good Fruit Vegetables Magaz 11:50.
Kivanc M, Akgul A, Dogan A (1991). Inhibitory and stimu- latory effects of cumin, oregano and their essential oils on growth and acid production of Lactobacillus plantarum and Leuconostoc mesenteroides. Internat J Food Microbiol 13:81-86.
Kumar A, Shukla R, Singh P, Prasad CS, Dubey NK (2008).
Assessment of Thymus vulgaris L. Essential oil as a safe bo- tanical preservative against postharvest fungal infestation of food commodities. Internat Food Sci Em Technol 9:575- 580.
Marino M, Bersani C, Comi G (1999). Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method. J Food Protect 62:1017-1023.
Martinez-Romero D, Guillen F, Valverde JM, Bailen G, Zapata P, Serrano M (2007). Influence of carvacrol on survival of Botrytis cinerea inoculated in table grapes. Internat J Food Microbiol 115:144-148.
Nigro F, Ippolito A, Lanttanzio V, Di Venere D, Salerno M (2000). Effect of ultraviolet-C light on postharvest decay of strawberry. J Plant Pathol 82:29-37.
Rolland F, Gonzalez E, Sheen J (2006). Sugar sensing and sig- naling in plants: Conserved and novel mechanisms. Ann Rev Plant Biol 57:675-709.
Saini RS, Sharma KD, Kaushik RA, Dhankhar OP (2006).
Laboratory Manual Analytical Techniques in Horticulture.
with concentration 100, 200 and 300 µl/l (Fig. 10). Be- cause polyethylene package performs as a barrier against water steam it condenses the water vapor in the pack- age atmosphere on the surface of grapes and leads to re- absorption of water. On the cool surface of the fruit due to high water absorption gaps appear in the cell walls that cause cracking of the berries skin (Considine and Kriede- mann, 1972). It seems high concentrations of essential oil led to reduction in water loss.
Quality of fruits
The quality of fruits (color, texture, aroma, taste and appearance of fruits) reduced along the storage (Tab. 2).
The control and treated samples with 100, 200 and 300 µl/l concentrationswere infected in the first 30 days.
Therefore, there was no data available after that. Quality of treated samples with 400 and 500 µl/l thyme essential oil evaluated until 45 days of storage. Treated samples preserved a better quality compared to controls (Fig. 11).
There was no significant difference among different con- centrations of essential oil in regard to quality. Except sam- ples which treated with 400 µl/l essential oil. They showed a significant decreased in quality of fruits. Essential oil did not have any adverse effect on aroma and taste of treated tomatoes and strawberries (Tzortzakis, 2007). Grapes that treated with thymol and eugenol had better quality com- pared to controls (Valero et al., 2006).
References
Adams RP (1995). Identification of essential oil components by Gas Chromatography/Mass Spectrometry. Allured Publish- ing Corporation, Carol Stream, Illinois.
Adisa VA (1986). The influence of molds and some storage fac- tors on the ascorbic acid content of orange and pineapple fruits. Food Chem 22:139-146.
Fig. 11. The effect of essential oil treatment on fruit quality of
‘Bidaneh Qermez’ table grape
Valero D, Valverde JM, Martinez-Romero D, Guillen F, Catioo S, Serrano M (2006). The combination of modified atmo- sphere packaging with eugenol or thymol to maintain qual- ity, Safety and functional properties of table grapes. Posthar- vest Biol Technol 41:317-327.
Wang CY, Buta JG (2003). Maintaining quality of fresh-cut ki- wifruit with volatile compound. Postharvest Biol Technol 28:181-186.
Xu WT, Huang KI, Guo F, Qu W, Yang JJ, Liang ZH, Luo YB (2007). Postharvest grapefruit seed extract and chitosan treatments of table grapes to control Botrytis cinerea. Post- harvest Biol Technol 46:86-94.
You-Mei WU, Jian-Chai REN, Xue-Zeng HUA, Yu LIU (2000).
Postharvest berry abscission and storage of grape fruit. Acad Scinica 32 p.
Agrobios (India). IST Edition, 12-26 p.
Shibamoto T (1987). Retention indices in essential oil analysis in capillary Gas Chromatography, 259-275 p. In: Essential oil analysis. Sandra P, Bicchic A (Eds.). Alfred Heuthig-Ver- lag. New York.
Sukata V, Haruthaithanosan V, Chntarapanont W, Dilokku- nanat V, Suppakul P (2008). Antifungol activity of clove and cinnamon oil and their synergistic against postharvest decay fungi of grape in vitro. Nat Sci 42:169-174.
Thumula P (2006). Studies on storage behavior of tomatoes coated with chitosan-lysozyme films. MSc Thesis. Faculty of Agricultural and Environmental Sciences. Mc Gill Univ Montreal, Quebec, Canada.
Tzortzakis NG (2007). Maintaining postharvest quality of fresh produce with volatile compounds. Internat Food Sci Em Technol 8:111-116.
Tzortzakis NG, Economakis CD (2007). Antifungal activity of lemon grass (Cympopogon citrates L.). Essential oil against key postharvest pathogens. Internat Food Sci Em Technol 8:253-258.
