Antioxidant and Antimicrobial Activities of Beet Root.pdf

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Czech J. Food Sci. Vol. 29, 2011, No. 6: 575–585 Antioxidant and Antimicrobial Activities of Beet Root Pomace Extracts Jasna M. Čanadanović-Brunet, Sladjana S. Savatović, Gordana S. Ćetković, Jelena J. Vulić, Sonja M. Djilas, Siniša L. Markov and Dragoljub D. Cvetković Faculty of Technology, University of Novi Sad, Novi Sad, Serbia Abstract Čanadanović-Brun
    575575 Czech J. Food Sci. Vol. 29, 2011, No. 6: 575–585 he association between the diet rich in fruits and  vegetables and a decreased risk of cardiovascular diseases and certain forms of cancer is supported by considerable epidemiological evidence (N & P 1999; R & N 2003). Different studies have shown that free radicals present in the human organism cause oxidative damage to  various molecules, such as lipids, proteins, and nucleic acids, and are thus involved in the initiation phase of the degenerative diseases. Phenolic and other phytochemical antioxidants found in fruits and vegetables are capable of neutralising free radicals and may play a major role in the preven-tion of certain diseases (K & K 2001).   Beet root (  Beta vulgaris  L. ssp. vulgaris , Che-nopodiaceae) ranks among the 10 most powerful  vegetables with respect to its antioxidant capacity ascribed to a total phenolic content of 50–60 μmol/g dry weight (V et al  . 1998; K et al  . 1999).   Beet root is a potential source of valuable water-soluble nitrogenous pigments, called beta-lains, which comprise two main groups, the red betacyanins and the yellow betaxanthins. hey are free radical scavengers and prevent active oxy-gen-induced and free radical-mediated oxidation of biological molecules (P & E 2001).   Betalains have been extensively used in the modern food industry. hey are one of the most Partly supported by the Ministry of Education and Science of the Republic of Serbia, Project No. 31044. Antioxidant and Antimicrobial Activities of Beet Root Pomace Extracts  J M. ČANADANOVIĆ󰀭BRUNE, S S. SAVAOVIĆ, G S. ĆEKOVIĆ,  J J. VULIĆ, S M. DJILAS, S L. MARKOV and D D. CVEKOVIĆ   Faculty of echnology, University of Novi Sad, Novi Sad, Serbia Abstract Čć-B J.M., Sć S.S., Ćć G.S., Vć J.J., D S.M., M S.L., Cć D.D. (2011): Antioxidant and antimicrobial activities of beet root pomace extracts . Czech J. Food Sci., 29 : 575–585. We described the in vitro  antioxidant and antimicrobial activities of ethanol, acetone, and water extracts of beet root pomace. otal contents of phenolics (316.30–564.50 mg GAE/g of dry extract), flavonoids (316.30–564.50 mg RE/g of dry extract), betacyanins (18.78–24.18 mg/g of dry extract), and betaxanthins (11.19–22.90 mg/g of dry extract) after solid-phase extraction were determined spectrophotometrically. Te antioxidant activity was determined by measuring the reducing power and DPPH scavenging activity by spectrometric metod, and hydroxyl and superoxide anion radical scavenging ac-tivity by ESR spectroscopy. In general, the reducing power of all the beet root pomace extracts increased with increasing concentrations. Te DPPH-free radical scavenging activity of the extracts, expressed as EC 50 , ranged from 0.133 mg/ml to 0.275 mg/ml. Significant correlation was observed between all phytochemical components and scavenging activity. 0.5 mg/ml of ethanol extract completely eliminated hydroxyl radical, which had been generated in Fenton system, while the same concentration of this extract scavenged 75% of superoxide anion radicals. In antibacterial tests, Staphylococcus aureus and   Bacillus cereus showed higher susceptibility than  Escherichia coli  and  Pseudomonas aeruginosa. Keywords :  Beta vulgaris  L.; phenolic compounds; bacterial culture; betalains; radical scavengig activity; electron spin resonance  576   576   Vol. 29, 2011, No. 6: 575–585 Czech J. Food Sci. important natural colorants and are also one of the earliest natural colorants developed for the use in food systems (F 1999; A 2009).   A more recent investigation showed that total phenolics content decreases in the order peel (50%), crown (37%), and flesh (13%). he peel also carries the main portion of betalains with up to 54%, their content being lower in crown (32%) and flesh (14%) (K et al  . 2000). Whereas the coloured fraction consists of betacyanins and be-taxanthins, the phenolic portion of the peel shows -tryptophane,  p -coumaric and ferulic acids, as well as cyclodopa glucoside derivatives (K et al  . 2001). Also, beet root contains a significant amount of phenolic acids: ferulic, protocatechuic,  vanillic,  p -coumaric,  p -hydroxybenxoi, and syringic acids. he high content of folic acid amounting to 15.8 mg/g dry matter is another nutritional feature of the beets (W  G 1997).On the industrial scale, juices are produced by pressing mashed fruits and vegetables, with or without the application of pectolytic enzyme preparations, using different equipment. Howe- ver, most of the secondary plant metabolites and dietary fiber compounds are not transferred into the liquid phase during the dejuicing process and remain in the pomace after pressing (W 2000). hough still rich in betalains and phenols, the beet root pomace from the juice industry (15–30%) is disposed of as feed and manure. hus new aspects, concerning the use of these wastes as by-products for further exploitation, are gaining an   increasing interest because these are high-value products and their recovery may be economically attractive (S 2001).In the food industry, synthetic antioxidants, such as butylated hydroxyanizole (BHA) and butylated hydroxytoluene (BH), have long been widely used as antioxidant additives to preserve and stabilise the freshness, nutritive value, flavour and colour of foods, and animal feed products. However, at least one study has revealed that BH could be toxic, especially at high doses (S et al  . 1995). Nowadays, there is an increasing interest in the substitution of synthetic food antioxidants by natural ones. he antioxidant compounds from waste products of food industry could be used for protecting the oxidative damage in living systems by scavenging oxygen free radicals, and also for increasing the stability of foods by preventing lipid peroxidation (M et al  . 2007). Special attention is focussed on their extraction from inexpensive or residual sources coming from ag-ricultural industries. he objectives of this study were: ( i ) to examine the phenolic and betalain compositions of beet root pomace extracts using spectrophotometrical determination of total phe-nolics, flavonoids, and betalains, ( ii ) to determine the antioxidant activity of pomace extracts on stable 2,2-diphenyl-1-picrylhydrazyl (DPPH ● ), reactive hydroxyl ( ● OH), and superoxide anion (O 2 ● – ) free radicals and the reducing power of the extracts obtained, (iii) to establish correlations between the phenolic and belalain compositions and antioxidant activity ( iv ) to determine the an-tibacterial activity. MATERIAL AND METHODS Chemicals . 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyroline-  N  -oxide (DMPO), KO 2 per crown ether, Folin-Ciocalteu reagent, BHA, chlorogenic acid and rutin were purchased from Sigma Chemical Co. (St. Louis, USA). hese chemicals were of analytical reagent grade. Other chemicals and solvents used were of the highest analytical grade and were obtained from Zorka, Šabac (Serbia).  Plant material  .   Beet root pomace were obtained from the factory for fruit and vegetable processing Zdravo organic, Selenca, Serbia.  Bacterial cultures .  Escherichia coli (ACC 10526),  Pseudomonas aeruginosa (ACC 27853), Staphylococcus aureus (ACC 11632), and  Bacillus cereus (ACC 10876) microorganism strains were employed for the determination of antimicrobial activity. he cultures of the test bacteria were grown 20–24 h in Müller-Hinton agar (orlak, Belgrade, Serbia) at 37°C and then transferred to Müller-Hinton broth. he inocula were prepared by adjusting the turbidity of the medium to match 5 × 10 6  CFU/ml.he antibiotic chloramphenicol used as refer-ence standard was obtained from Sigma-Aldrich (Steinheim, Germany). Bacteria were obtained from the stock cultures of Microbiology Laboratory, Faculty of echnol-ogy, University of Novi Sad.  Extraction procedure . he samples of beet root pomace (40 g) were extracted at room temperature for 2 × 24 hours. he extraction was performed with different solvents: 80% ethanol aqueous solu-tion containing 0.5% acetic acid (E1), 80% acetone    577577 Czech J. Food Sci. Vol. 29, 2011, No. 6: 575–585 aqueous solution containing 0.5% acetic acid (E2), and 0.5% acetic acid in water (E3). he extracts obtained were combined and evaporated to dryness under reduced pressure. he yields of extracts were: m E1  = 3.32 g; m E2  = 3.09 g, and m E3  = 3.55 g.  Extract purification .   A solid-phase extraction (SPE) with a vacuum manifold processor (system spe-12G; J.. Baker, Großgerau, Germany) with CHROMABOND C 18  column (1000 mg; J.. Ba-ker, Phillipsburg, USA) was used for the extract purification in order to remove the organic acids, residual sugars, amino acids, proteins, and other hydrophilic compounds (R et al  . 2000). he CHROMABOND C 18  column was preconditioned by passing 8 ml of methanol and 20 ml of 5mM H 2 SO 4 . Beet root pomace extract was dissolved in 10 ml of 0.5M H 2 SO 4 . he extract solution was loaded onto the preconditioned column. he co-lumn was washed with 8 ml of 5mM H 2 SO 4 . he purifed extract was eluted with 8 ml of methanol and 20 ml of distiled water. he purified beet root extract was evapored to dryness under reduced pressure. he obtained weights of SPE-purified extracts were: m pE1  = 0.130 g; m pE2  = 0.085 g and m pE3  = 0.128 g. Total phenolic content  . he amount of total soluble phenolics in the extracts was determined spectrophotometrically according to the Folin-Ciocalteu method (S et al  . 1999). he reaction mixture was prepared by mixing 0.1 ml of water solution (concentration 1 mg/ml) of the extract, 7.9 ml of distilled water, 0.5 ml of Folin-Ciocalteu’s reagent and 1.5 ml of 20% sodium carbonate. After 2 h, the absorbance at 750 nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan) was read against control that had been prepared in a similar manner, by replacing the extract with distilled water. he total phenolic content, expressed as mg of gallic acid equivalents (GAE) per g of dry beet root pomace extract, was determined using the calibration curve of gallic acid standard. Total flavonoids .  otal flavonoids were deter-mined using the colorimetric assay developed by Z et al  . (1999). An aliquot (1 ml) of extract (concentration 1 mg/ml) was added to 10 ml volu-metric flask containing 4 ml of distilled H 2 O. Into the flask, 0.3 ml 5% NaNO 2  was added and 5 min later 0.3 ml 10% AlCl 3  was added. After 6 min, 2 ml of 1M NaOH solution was added and the total  volume was made up to 10 ml with distilled H 2 O. he solution was well mixed and the absorbance was measured at 510 nm against the control that had been prepared in the same manner only with replacing the extract with distilled water. otal flavonoid content was expressed as mg rutin equi- valents (RE) per g of dry extract. Total betalain content  . he total betalain (be-tacyanin and betaxanthin) pigment content in the extract was measured spectrophotometrically. he wavelengths of 535 nm and 476 nm were used for betacyanin and betaxanthin analysis, respective-ly ( E 2003). he aqueous extracts were diluted with 0.05M phosphate buffer (pH 6.5) to obtain the absorption values A 538  of 0.4 < A < 0.5. he absorbance was read at 476, 538, and 600 nm. he calculations were carried out according to the following equations:  x  = 1.095 ( a – c );  y  = b  – z   –  x /3.1; and z   = ax , where: a = absorbance at 538 nm; b  = absorbance at 476 nm; c  = absor-bance at 600 nm;  x  = absorbance of betacyanin;  y  = absorbance of betaxanthin; and z   = absorbance of impurities. he contents of betacyanin (BC) and betaxanthin (BX) were calculated using the equations: BC (g/100 ml) = 1000 ×  x  × F/A 1%,betanin BX (mg/100 ml)= 1000 ×  y  × F/A 1%,vulgaxanthin-I where: F – dilution factorA 1%,vulgaxanthin-I , A 1%,betanin  – extinction coefficients repre-senting the absorption of 1% solution (1 g/100 ml), being 1120 for betanin and 750 for vulgaxanthin-I otal betacyanin content was expressed as mg of betanin equivalents per g of dry extract, and total betaxanthin content was expressed as mg of  vulgaxanthin-I equivalents per g of dry extract. 2,2-diphenyl-1-picrylhydrazyl radical scaven- ging activity  . he free radical scavenging activity of the extracts was determined spectrophotome-trically. he hydrogen atom or electron donation abilities of the extract was measured from the bleaching of a purple-coloured methanol solution of stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ● ). Briefly, 1 ml of solution containing from 0.1 to 0.5 mg of extract in 95% methanol or 1 ml of methanol (control) were mixed with 3 ml of 90μM DPPH solution (18 mg in 50 ml 95% met-hanol prepared daily) and 8 ml of 95% methanol. he mixture was vortexed thoroughly for 1 min and left at room temperature for 60 min, than the absorbance was read against control at 515 nm.  578   578   Vol. 29, 2011, No. 6: 575–585 Czech J. Food Sci. he control probe contained all components except the radicals. he capability to scavenge the DPPH radicals, DPPH ●  scavenging activity (SA  DPPH ● ), was calculated using the following equation: SA DPPH ● (%) = (A control  – A sample )/A control  × 100 where: A control  – absorbance of the control reaction (containing all reagents except the extract)A sample  – absorbance in the presence of the extract  Reducing power  . he reducing power of the extracts was determined by the method of O (1986). he capacity of the extracts to reduce the ferric-ferricyanide complex to the ferrous-ferri-cyanide complex of Prussian blue was determi-ned by recording the absorbance at 700 nm after incubation. For this purpose, the suspensions of extracts (0.05–1 mg) in 1 ml of distilled water or 1 ml of distilled water (control) were mixed with 1 ml of phosphate buffer (pH 6.6) and 1 ml of 1% potassium ferricyanide K 3 [Fe(CN) 6 ]. he mixture was incubated at 50°C for 20 minutes. Following this, 1 ml of trichloroacetic acid (10%) was added and the mixture was then centrifuged at 3000 rpm for 10 minutes. A 2 ml aliquot of the upper layer was mixed with 2 ml of distilled water and 0.4 ml of 0.1% FeCl 3 , and the absorbance of the mixture was measured at 700 nm. he absorbance at 700 nm was used as the indicator of the reducing power. Increased absorbance of the reaction mixture in-dicated increased reduction capability. Ascorbic acid was used as positive control.  Hydroxyl radical scavenging activity  .   As hyd-roxyl free radicals ( ● OH) are highly reactive, with relatively short half-lives, the concentrations found in natural systems are usually inadequate for the direct detection by ESR (electron spin resonance) spectroscopy. Spin-trapping is a chemical reaction that provides an approach to help overcome this problem. Hydroxyl radicals are identified by means of their ability to form nitroxide adducts (stable free radicals form) from the commonly used DMPO (5,5-dimethyl-1-pyroline-  N  -oxide) as the spin trap. he Fenton reaction was conducted by mixing 0.2 ml of 0.3M DMPO, 0.2 ml of 10mM H 2 O 2 , and 0.2 ml of 10mM Fe 2+  (control). he influence of different ethanol extracts on the formation and stabilisation of hydroxyl radicals was investigated by adding the investigated extracts in the Fenton reaction system in the concentration range 0.01–0.5 mg/ml. ESR spectra were recorded after 5 min, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.512 G, receiver gain 5 × 10 5 , time constant 81.92 ms, conversion time 327.68 ms, center field 3,440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23°C.he SA ● OH  value of the extract was defined as: SA ● OH  = 100 × (h 0  – h x )/h 0  (%) where:h 0 , h x  – heights of the 2 nd  peak in the ESR spectrum of DMPO-OH spin adduct of the control and the probe Superoxide anion radical scavenging activity  . Superoxide anion radicals (O 2 ● – ) were generated in the reaction system obtained by mixing 500 µl of dry dimethylsulfoxide (DMSO), 5 µl of KO 2 /crown ether (10mM/20 mM) prepared in dry DMSO, and 5 µl of 2M water solution of DMPO as the spin trap. Te influence of the ethanol extracts on the formation and transformation of superoxide anion radicals was determined by adding dimethylforamide (DMF) solutions of the beet root extract fractions to the superoxide anion reaction system in the final range of concentrations 0.01–1 mg/ml (probe). After that, the mixture was stirred for 2 min and transferred to a quartz flat cell ER-160F. Te ESR spectra were recorded on an EMX spectrometer from Bruker (Rheinstetten, Germany) under the following con-ditions: field modulation 100 kHz, modulation am-plitude 4.00 G, receiver gain 1 × 10 4 , time constant 327.68 ms, conversion time 40.96 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW and temperature 23°C. he SAo 2 ● –  value of the extract was defined as: SAo 2 ● –  = 100 × (h 0  – h  x )/h 0  (%) where:h 0 , h  x  – heights of the 2 nd  peak in the ESR spectrum of DMPO-OOH spin adduct of the control and the probe  Antibacterial activity  . For the evalution of the antibacterial activity of the samles, agar disc dif-fusion method was used. he strains were grown on Mueller-Hinton agar slants at 37°C for 24 h and checked for purity. After the incubation, the cells were washed off the surface of agar and suspended in sterile physiological solution. he number of cells in 1 ml of suspension for inoculation measured by McFarland nefelometer was 5 × 10 7  CFU/ml.
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