© Borgis - Postępy Fitoterapii 2/2013, s. 85-89
*Agata Stobnicka1, Elvira Jungfer2, Małgorzata Gniewosz1
Skład i właściwości antybakteryjne świeżego soku z żurawiny wielkoowocowej (Vaccinium macrocarpon L.)
Composition and antibacterial properties of fresh cranberry (Vaccinium macrocarpon L.) juice**
1Department of Biotechnology, Microbiology and Food Evaluation, Warsaw University of Life Sciences, Poland
Head of Department: prof. Małgorzata Gniewosz, PhD
2Department of Nutrition and Food Sciences, University of Bonn, Germany
Head of Department: prof. Rudolf Galens
Celem niniejszych badań było określenie składu i właściwości antybakteryjnych surowego soku żurawinowego (Vaccinium macrocarpon L.). Działanie soku zostało sprawdzone w stosunku do bakterii typowych dla żywności (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922 i Salmonella enteritidis ATCC 13076). Badany sok wykazał aktywność przeciwbakteryjną wobec wszystkich szczepów testowych. Najbardziej wrażliwy na jego działanie okazał się szczep Staphylococcus aureus ATCC 25923 (MIC = 2,3 mg/ml), a najbardziej oporny szczep Salmonella enteritidis ATCC 13076 (MIC = 19,2 mg/ml). Analiza składu soku metodą HPLC-UVDAD-MSn wykazała obecność m.in. procyjanidyn (dimery, trimery, tetramery), epikatechiny, kwasu chlorogenowego, glikozydów myrycetyny i kwercetyny. Wyniki te są obiecujące i dają nadzieję na potencjalne wykorzystanie soku żurawinowego w leczeniu zatruć pokarmowych i biegunki.
Berry fruits are rich sources of bioactive compounds such as phenolic and organic acids which may hold antimicrobial activities (1). Cranberry (Vaccinium macrocarpon L.) is a polyphenolic-rich berry fruit native to North America, but also grown and popularly used in traditional folk medicine in Europe as a treatment of microbial infections. Cranberry fruits are important and willingly eaten ingredient of the diet as well, which offers important health benefits (2-4).
Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella enteritidis are typical foodborne bacteria, which can cause food spoilage, food poisoning, stomach dysfunction and diarrhea. According to Tayel et al. (5), gastrointestinal disorders can be successfully treated with as conventional and as natural methods. However, consumers today are increasingly concerned not only about chemical residues in food, but also about side effects of chemical drugs and tend to choose natural food and natural methods of treatment, as well (6). Consequently development of natural antimicrobial compounds is on the rise. The objectives of the present research were to study (1) the antibacterial properties, including the bacteriostatic and bactericidal activity, of cranberry juice (Vaccinium macrocarpon L.) and HPLC-UVDAD-MSn analysis (2) of the juice sample.
Materials and methods
Fruits of Vaccinium macrocarpon L. („Pilgrim”) (Ericaceae) were collected in Poland, (52.002°N, 20.937°E).
Preparing of the juice
Cranberry fruits were pressed in Omega 8006 juicer (Omega, USA). The juice was filtrated through Whatman paper No.1. The filtrate was stored in refrigerator at 4°C till further use.
Reference bacterial strains were the Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) and the Gram-negative (Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076). All tested strains were obtained from American Type Culture Collection. All tested microorganisms were subcultured into Mueller-Hinton Agar (Btl, Poland) slants and were maintained at 4°C.
The antibacterial assay was performed using the broth macrodilution method (7). A twofold serial dilution of the juice was prepared in Mueller-Hinton Broth (MHB) (Merck, Germany). For every experiment a negative control (distilled water, medium, inoculums) was included. Each test-tube received 2 ml of the medium, 10 μl of the inoculum (106 CFU/ml) and in sequence a proper amount of the juice (2000 μl; 1000 μl; 500 μl; 250 μl; 125 μl; 60 μl; 30 μl; 15 μl). Mentioned volumes of the juice correspond with following dry mass content: 38 mg; 19 mg; 9,6 mg; 4,8 mg; 2,4 mg; 1,2 mg; 0,6 mg; 0,3 mg. The content of each tube was mixed thoroughly with tube shaker and incubated at 37°C for 24 h. Microbial growth in each test-tube was determined by observing and comparing the test-tube with the negative control. The absence of microbial growth was interpreted as an antibacterial activity. The minimum inhibitory concentration (MIC) was the lowest concentration of the juice that prevented visible growth of bacteria. Minimum bactericidal concentration (MBC) was determined by plating 100 μl from each negative and the first positive growth test-tube on Mueller-Hinton Agar (Btl, Poland). Plates were incubated at 37°C for 24 h. MBC was defined as the lowest concentration yielding 0-50 colonies on the plate.
To avoid any interference into cranberry juice, the influence of pH on bacteria’s growth was checked by adjusting pH of Mueller-Hinton Broth (MHB) (Merck, Germany), by the addition 0,1 M HCl, to the value detected by pH meter (Emerson, USA) in MIC tube. Then test-tube received 2 ml of the medium and 10 μl of the inoculum (106 CFU/ml). The content of each tube was mixed thoroughly with tube shaker and incubated at 37°C for 24 h. Microbial growth in test-tube was determined by observing and comparing the test-tube with the negative control.
Juice sample preparation with solid-phase extraction
Juice sample for analysis was prepared using the following solid-phase extraction (SPE) performed by using a Gilson ASPEC XL system (Automated Sample Preparation with Extraction Cartridges, Abimed, Langenfeld, Germany). Firstly, HR-XC SPE cartridge (500 mg, 3 ml cartridge, Macherey-Nagel, Düren, Germany) was conditioned with methanol and washed with UHQ water. The cartridge was loaded with 10 ml juice and washed with 0.1 n HCl. The polyphenols were eluted with 3 ml methanol.
The analysis of the phenolic compounds by HPLC-UVDAD-MSn was performed according to a method of Papagiannopolus et al. (8). The liquid chromatograph was a Summit system (Dionex, Idstein, Germany) consisting of a Degasys DG-1310 degasser (Uniflows, Tokyo, Japan), a P-580 A HPG pump, an ASI-100 T automated sample injector, an STH-585 column oven, and a UltiMate 3000 Diode Array Detector. Chromeleon software package Version 6.20 Build 531 was used for system control and data evaluation. Separation was carried out with an analytical column Nucleodur C18 Isis, 150 x 2 mm, 3 μm (Macherey-Nagel, Düren, Germany) temperated at 35°C. Solvents were UHQ water with 1 % acetic acid (v/v) (mobile phase A) and acetonitrile with 1% acetic acid (v/v) (mobile phase B). The injection volume was 10 μl and the chromatogram was monitored at 200-600 nm.
An LCQ Classic ion trap mass spectrometer equipped with an electrospray interface was coupled with the HPLC and controlled with Xcalibur software. All polyphenols were analysed in the negativ ion mode with following settings: source voltage,- 4.0 kV; sheath gas flow, 90; auxiliary gas flow, 60; capillary voltage, -10 V; capillary temperature, 300°C; first octapole offset, +4.0 V; interoctapole lens voltage, +30.0 V; second octapole offset, +10.0 V; ion trap DC offset, +10.0 V.
Polyphenols were identified using the UV spectral data and the molecular weight and fragmentation pattern from the mass spectrometer. Identification of individual compounds was conducted by comparison of characteristic mass fragmentation patterns with literature.
Results and discussion
Antibacterial activity of cranberry juice
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