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© 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
Streszczenie
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.
Słowa kluczowe: sok żurawinowy, Vaccinium macrocarpon, aktywność przeciwbakteryjna
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Introduction
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
Plant material
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.
Microorganisms
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.
Antibacterial assay
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.
HPLC-UVDAD-MSn analysis
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
In the present study, the cranberry juice (V. macrocarpon) showed the antibacterial activity against both Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram-negative (Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076) strains. Gram-negative bacteria were less sensitive than Gram-positive bacteria. The results summarized in table 1 demonstrate that the cranberry juice had the strongest antibacterial activity against Staphylococcus aureus ATCC 25923 (2.3 mg dry mass/ml) and the weakest against Salmonella enteritidis ATCC 13076 (19.0 mg dry mass/ml). MIC value for Bacillus subtilis ATCC 6633 was 4.8 mg dry mass/ml and for Escherichia coli – 9.6 mg dry mass/ml. Minimal bactericidal concentration had equal values for both Gram-positive strains (4.8 mg dry mass/ml) and for both Gram-negative strains (19.0 mg dry mass/ml).
Table 1. Inhibition parameters (MIC,MBC) of the cranberry (Vaccinium macrocarpon L.) juice.
MicroorganismsParameters
MIC
(mg d.m./ml) (medium pH)
MBC
(mg d.m./ml)
Staphylococcus aureus ATCC 259232.3 (6.96)4.8
Bacillus subtilis ATCC 66334.8 (6.20)4.8
Escherichia coli ATCC 259229.6 (4.98)19.0
Salmonella enteritidis ATCC 1307619.0 (4.26)19.0
d.m. – dry mass
The influence of pH on strains growth were summarized in table 2. Cranberry juice inhibited growth of tested strains (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922) at pH = 6.96, pH = 6.20 and pH = 4.98. As the results of checking influence of pH medium on bacterial growth showed, strain’s growth was observed at mentioned pH. Strong antibacterial properties on these strains are probably not result of low pH medium, but come out from influence of substances other than acids, like quercetin, myricetin and procyanidins (9). However, inhibition of Salmonella enteritidis ATCC 13076 growth at pH = 4.26, was especially result from low pH medium.
Table 2. Influence of pH medium on bacteria’s growth.
MicroorganismsMedium pHGrowth
Staphylococcus aureus ATCC 259236.96+
Bacillus subtilis ATCC 66336.20+
Escherichia coli ATCC 259224.98+
Salmonella enteritidis ATCC 130764.26
+ normal growth, – no growth
The results are in accordance to Côte et al. (10), who finds cranberries may offer a line of defense against food poisoning with their ability to reduce the growth of Salmonella sp., Escherichia coli, Staphylococcus aureus and other types of bacteria found in food. Cranberry juice has also been reported to inactivate other foodborne pathogens such as Listeria monocytogenes, Escherichia coli O157:H7 and Vibrio parahaemolyticus (11-14). As a result it is anticipated that phytochemicals with adequate antibacterial efficacy can be used for the treatment of the bacterial infections (15). Cranberry juice is known to exhibit a range of antibacterial, anti-viral and pharmacological activities (16-18). According to Magariños et al. (19) Staphylococcus aureus ATCC 25923 was the most susceptible to cranberry juice inhibition among all tested strains.
Composition of cranberry juice
The tentatively identified compounds are shown in table 3 and figure 1 shows the corresponding chromatogram monitored at 280 nm. As already described by Määttä-Riihinen et al. (20) and Gu et al. (21) epicatechin, procyanidin dimers (A- and B-type), trimers and tetramers could be shown in the cranberry juice. Different flavonols are detectable in the juice. According to Vvedenskaya et al. (22) the major flavonol glycoside in cranberries is quercetin as also show in this study and as well myricetin glycosides could be detected. The presence of the coumaroyl iridoid shown in peak 13 was already reported by Jensen et al. (23). In the group of phenolic acids caffeoyl hexoside (peak 2), chlorogenic acid (peak 3), and feruloyl quinic acid (peak 9) could be tentatively assigned. This is in accordance to Häkkinen et al. (24).
Table 3. Composition of the cranberry (Vaccinium macrocarpon L.) juice.
Peak No.Tentatively identified compoundRT (min)[M-H]-
1Procyanidin tetramer13.921152
2Caffeoyl hexoside17.5341
3Chlorogenic acid18.53353
4Procyanidin dimer19.19575
5Epicatechin 25.18289
6Unknown compound26.16
7Procyanidin trimer32.42863
8Myricetin hexoside38.18479
9Feruloyl quinic acid40.15367
10Procyanidin trimer41.08863
11Quercetin hexoside48.51463
12Procyanidin trimer50.35863
13Coumaroyl iridoid51.57537
14Quercetin pentoside53.5433
15Procyanidin trimer54.73863
16Quercetin pentoside55.39433
17Quercetin pentoside57.45433
18Myricetin59317
19Quercetin deoxyhexoside60.37447
20Dimethoxymyricetin hexoside63.25507
Fig. 1. HPLC Chromatogram of the SPE extract at 280 nm.
Conclusions
In conclusion, the present study demonstrates that cranberry juice (V. macrocarpon) has antibacterial effects against S.aureus, B. subtilis, E.coli and S. enteritidis and contributes to the knowledge of the natural antibacterial substances. The results provide promising baseline information for the potential use of cranberry juice for food poisoning and diarrhea treatment and may be further investigated as a natural solution for gastrointestinal disorders.
Acknowledgements
The authors would like to thank the owners of “Grąbczewscy – nursery garden since 1936” for their contribution to this work.

**Project cofinanced by the European Union under the European Social Fund, Project No. 897.
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Piśmiennictwo
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otrzymano: 2013-01-23
zaakceptowano do druku: 2013-02-05

Adres do korespondencji:
*Agata Stobnicka
Department of Biotechnology, Microbiology and Food Evaluation Warsaw University of Life Sciences
ul. Nowoursynowska 159, 02-776 Warszawa
tel.: +48 (22) 593-76-58, fax: + 48 (22) 593-76-81
e-mail: agatastobnicka@gmail.com

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