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Journal of Food Protection, Vol. 78, No. 6, 2015, Pages 1186–1190 doi:10.4315/0362-028X.JFP-14-440 Copyright G, International Association for Food Protection

Research Note Adhesion of bogorensis to Glass and Polystyrene in the Presence of Cranberry Juice

HUBERT ANTOLAK,* DOROTA KREGIEL, AND AGATA CZYZOWSKA

Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland

MS 14-440: Received 10 September 2014/Accepted 29 January 2015 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/78/6/1186/1686447/0362-028x_jfp-14-440.pdf by guest on 02 October 2021 ABSTRACT The aim of the study was to evaluate the adhesion abilities of the acetic acid bacterium to glass and polystyrene in the presence of American cranberry (Vaccinium macrocarpon) juice. The strain of A. bogorensis used was isolated from spoiled commercial fruit-flavored drinking water. The cranberry juice was analyzed for polyphenols, organic acids, and carbohydrates using high-performance liquid chromatography and liquid chromatography–mass spectrometry techniques. The adhesive abilities of bacterial cells in culture medium supplemented with cranberry juice were determined using luminometry and microscopy. The viability of adhered and planktonic bacterial cells was determined by the plate count method, and the relative adhesion coefficient was calculated. This strain of A. bogorensis was characterized by strong adhesion properties that were dependent upon the type of surface. The highest level of cell adhesion was found on the polystyrene. However, in the presence of 10% cranberry juice, attachment of bacterial cells was three times lower. Chemical analysis of juice revealed the presence of sugars, organic acids, and anthocyanins, which were identified as galactosides, glucosides, and arabinosides of cyanidin and peonidin. A-type proanthocyanidins responsible for the antiadhesion properties of V. macrocarpon also were detected.

Red cranberries (Vaccinium macrocarpon Ait., family to human erythrocytes and human Ericaceae) are native to North America. Cranberries were gastric mucous (3) and can inhibit coaggregation and reduce first used by Native Americans, who discovered the wild salivary counts of oral (24). The antiadhesive berry’s versatility as a food, fabric dye, and healing agent. properties of cranberry can affect various microorganisms The name ‘‘cranberry’’ derives from ‘‘craneberry,’’ the (20), but less attention has been paid to cranberry juice as an name used by early European settlers in America because antiadhesive and antibacterial agent against acidophilic or the small pink or red blossoms resemble the head and bill of acid-tolerant food spoilage bacteria. a crane. Today, cranberries are used for juices, cocktails, The aims of this study were to investigate the effects of jellies, and sauces. cranberry juice on the food-spoiling acetic acid bacterium Scientific studies have revealed that cranberry com- Asaia bogorensis. Many Asaia share the ability to pounds have potential health effects against cancer, aging coaggregate, forming both flocks in beverages and biofilms and neurological diseases, inflammation, diabetes, and on production surfaces (11–13). These biofilms are very bacterial infections (18). The cranberry also has been stable and difficult to remove using standard cleaning and recognized for its contribution to maintenance of a healthy disinfection methods (25). The type of food packaging used urinary tract. Although the earliest studies suggested that can affect the development of spoilage microflora (8). acidification of urine was responsible for the cranberry’s Polystyrene and glass are packaging materials commonly effect on urinary tract health, research since the 1980s has used in the beverage industry (1, 22). We studied the ability focused on the specific antiadhesive properties of cranberry of A. bogorensis cells to adhere to two carriers, glass and juice. Zafriri and coworkers (29) were the first to postulate polystyrene, in the presence of 10% (vol/vol) cranberry that various compounds in cranberries could affect P and juice. This work is the first to describe the use of cranberry type 1 fimbriae of , and they found that juice as a natural antiadhesive agent against the acidophilic fructose inhibited the adhesion of bacteria with type 1 food spoilage bacteria Asaia spp. fimbriae. In 1998, Howell et al. (10) identified specific proanthocyanidin compounds in cranberries that caused P- MATERIALS AND METHODS fimbriated E. coli to exhibit antiadhesion properties, and the Bacterial cultures. A. bogorensis strain FFMW (GenBank chemical structure of these compounds was later described KC756841.1) was isolated from flavored mineral water in Poland, (6). Cranberry juice inhibits the adhesion of three strains of and polyphasic identification was based on both physiological and genetic testing (12, 13). For aerobic bacterial cultures, commercial * Author for correspondence. Tel: z48426313475; Fax: z48426365976; fruit-flavored mineral water was used as the liquid culture medium: E-mail: [email protected]. 8.1% saccharose (wt/vol), 0.05% strawberry flavor (wt/vol), J. Food Prot., Vol. 78, No. 6 CRANBERRY JUICE INHIBITS A. BOGORENSIS ADHESION 1187 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/78/6/1186/1686447/0362-028x_jfp-14-440.pdf by guest on 02 October 2021

FIGURE 1. Chromatographic profiles of cranberry juice for acids and saccharides (A) and polyphenols (B). (A) Peak 1, citric acid; peak 2, glucose; peak 3, malic acid; peak 4, fructose and quinic acid. (B) Peak 1, cyanidin-3-galactoside; peak 5, cyanidin-3-arabinoside; peak 7, peonidin-3-galactoside; peak 8, peonidin-3-glucoside; peak 11, peonidin-3-arabinoside.

0.16% citric acid (wt/vol), 0.02% sodium benzoate (wt/vol), and Carriers. Polystyrene slides (76 by 26 mm; Packor 0.02% velcorin (wt/vol), pH 3.5. The fruit-flavored mineral water Packaging, Skierniewice, Poland) were used as sample carriers. was sterilized by membrane filtration (0.45-mm-pore-size filter; This material is approved by the Polish National Institute of Public Millipore, Billerica, MA). The culture medium (20 ml) was poured Health for contact with food. White glass slides (76 by 26 mm; Star into a 25-ml Erlenmeyer flask, and sterile sample carriers were Frost, Knittel Glass, Braunschweig, Germany) were used as the placed vertically so that half of the carrier was immersed in reference material. medium, leaving the other part suspended over the medium. Culture media with and without cranberry juice (10%, vol/vol) Adhesion analysis. The amount of inoculum was standard- were used. Two types of medium contained cranberry juice: (i) ized to obtain levels in culture medium of approximately 104 CFU/ without pH correction and (ii) with pH correction using 0.1 M ml at the beginning of the experiment. The samples were incubated NaOH. at 25uC on a laboratory shaker (130 rpm) for 6 days. Bacterial growth and adhesion in the presence of cranberry juice were Cranberry juice. Cranberries (V. macrocarpon) were analyzed by comparing results obtained after 0, 3, and 6 days of purchased from a local retail market (Lodz, Poland). The fresh incubation. The results of experiments without cranberry supple- fruits were washed with sterile water, lightly dried with a sterile mentation were used as the control. The analysis of adhesion to the paper towel, frozen at 220uC, and stored frozen for 3 months. carriers was done by luminometry and the plate count method. For After defrosting, the fruits were pressed with a juice extractor luminometric tests, the carrier plate was removed from the culture (MES3000, Bosch, Warsaw, Poland). The cloudy juice was filtered medium, washed with sterile distilled water, and swabbed with with a qualitative paper filter (Whatman, Clifton, NJ) and then with pens for sampling free ATP (Merck, Darmstadt, Germany). a sterile 0.45-mm-pore-size membrane filter (Millipore). Measurement was reported in relative light units (RLU) from a 1188 ANTOLAK ET AL. J. Food Prot., Vol. 78, No. 6

FIGURE 2. Adhesion of Asaia bogorensis to glass and polysty- rene in flavored mineral water (light shading), flavored mineral Downloaded from http://meridian.allenpress.com/jfp/article-pdf/78/6/1186/1686447/0362-028x_jfp-14-440.pdf by guest on 02 October 2021 water with cranberry juice (medium shading), and flavored mineral water with cranberry juice after pH correction (dark shading). Data were analyzed with a one-way repeated measures ANOVA. * P , 0.05; ** P , 0.01; *** P , 0.001 compared with the control medium (flavored mineral water).

HY-LiTE2 luminometer (Merck). For the determination of viable bacterial cells, both on the tested surface and in the culture medium, the colony method was used. For the determination of the number of attached bacterial cells, previously removed biofilm was vortexed in 0.85% saline with 0.1% Tween 80, and dilutions were transferred onto GC agar (2% D-glucose, 0.3% peptone [wt/vol], 0.3% yeast extract [wt/vol], and 0.7% CaCO3 [wt/vol]). After incubation (25uC for 96 h), the Asaia colonies were counted, and FIGURE 3. Relative adhesion coefficient A of Asaia bogorensis the number of attached bacterial cells per square centimeter of the calculated for glass and polystyrene in flavored mineral water carrier was determined. Corresponding medium and conditions (light shading), flavored mineral water with cranberry juice were used for the determination of the number of planktonic cells (medium shading), and flavored mineral water with cranberry per cubic centimeter of the medium. The relative adhesion juice after pH correction (dark shading). Data were analyzed with coefficient (A) was calculated using the formula A ~ (N /N ) | a p a one-way repeated measures ANOVA. * P , 0.05; ** P , 0.01; 100, where N is the number of cells attached to the carrier and N a p *** P , 0.001 compared with the control medium (flavored is number of planktonic cells (11). mineral water). Chemical constituent analysis. Organic acid and carbohy- 96% solvent A for 2 min, followed by washing and re-equilibration drate profiles of cranberry juice were determined using high- of the column. Mass spectra were recorded within 60 min. The performance liquid chromatography (HPLC) (7). Anthocyanins injection volume was 10 ml. The flow rate was set at 220 ml/min. were identified using HPLC with a diode array detector (Finnigan Electrospray ionization mass spectrometry was performed using the Surveyor-PDA Plus, Thermo Fisher Scientific, Waltham, MA) and LTQ Velos MS (Thermo Fisher Scientific) equipped with a heated ChromQuest 5.0 chromatography software (Thermo Fisher Scien- electrospray ionization interface and controlled by Excalibur tific). Separation was achieved on a Lichrospher RP 18-5 (250 by software (Chattanooga, TN). Mass spectra were acquired in negative 4.6 mm, 5-mm packing; Hichrom, Reading, UK). The elution mode over the m/z range of 120 to 1,000. The ionization spray conditions were as follows: flow rate of 0.8 ml/min; oven voltage was 4 kV. The sheath gas flow rate was 25, and the aux gas temperature of 25 C; solvent A, water:formic acid (95:5, vol/ u flow rate was 10. The temperatures of source and desolvation were vol); and solvent B, 95% acetonitrile. Elution began with 3% 350 and 280uC, respectively. solvent B for 2 min, then 3 to 15% solvent B for 13 min, 15 to 18% solvent B for 9 min, 18 to 25% solvent B for 31 min, and 25 Statistical analysis. Means were calculated from the data to 30% solvent B for 5 min, followed by washing and re- obtained from the three independent experiments, and the standard equilibration of the column. The injection volume for all samples deviation (SD) was calculated. Comparisons between the mean was 50 ml. Detection was conducted at 520 nm. Other polyphenols values of adhesion results were performed using a one-way were characterized using liquid chromatography–mass spectrometry repeated measures analysis of variance (ANOVA; OriginPro (LC-MS; LTQ Velos MS, Thermo Fisher Scientific). Chromato- 9.2.214, OriginLab Corp., Northampton, MA). Statistical signifi- graphic separation was achieved with a column operated at 45uC. cance was set at the conventional level of 5% (P , 0.05). The mobile phase consisted of solvent A (1 ml of formic acid in 1 liter of deionized water) and solvent B. The column used was a RESULTS AND DISCUSSION Hypersil Gold (150 by 2.1 mm, particle size of 1.9 mm; Thermo Fisher Scientific). The elution began with 96 to 85% solvent A for Figure 1 includes chromatograms of compounds de- 8 min, then 85 to 82% solvent A for 12 min, 82 to 60% solvent A for tected in cranberry juice and a table with the concentrations 40 min, 60 to 50% solvent A for 4 min and then 3 min, and 50 to of the individual sugars, organic acids, and polyphenols. J. Food Prot., Vol. 78, No. 6 CRANBERRY JUICE INHIBITS A. BOGORENSIS ADHESION 1189

4 7 6 3 7 7 4 7 8 Cranberry juice contained fructose and glucose as main 10 10 10 10 10 10 10 10 10 saccharides and citric, malic, and quinic acids. The five | | | | | | | | |

) major anthocyanins were identified as galactosides, gluco- 3 1.7 1.0 9.9 4.6 1.5 5.4 1.2 3.2 1.4 sides, and arabinosides of cyanidin and peonidin. The

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ presence of polyphenols and organic acids was also 4 7 7 4 7 7 4 8 8 (CFU/cm

10 10 10 10 10 10 10 10 10 confirmed by LC-MS. Other compounds detected during Planktonic cells

| | | | | | | | | the analysis were ferulic acid, gallic acids, chlorogenic and neochlorogenic acids, quercetin-3-rhamnoside, quercetin-3- 5.9 1.1 2.4 2.2 2.0 1.1 2.1 1.6 4.3 glucuronide, quercetin-3-benzylogalactoside, and methyl-

3 6 6 1 5 6 2 6 6 quercetin-3-xyloside. We also detected type A proantho-

10 10 10 10 10 10 10 10 10 cyanidins, which are responsible for antiadhesive properties

| | | | | | | | | of V. macrocarpon (5, 9). ) 2 Polystyrene 2.6 4.4 7.2 1.7 3.6 1.8 2.8 3.4 7.2 Figures 2 and 3 present the results of adhesion studies

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ after 6 days of incubation. Biofilm formation was dynamic 3 6 7 2 6 6 2 6 7 (CFU/cm and changeable. Adhesion of A. bogorensis cells was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/78/6/1186/1686447/0362-028x_jfp-14-440.pdf by guest on 02 October 2021 10 10 10 10 10 10 10 10 10 Adhered cells significantly higher on polystyrene. After 6 days of | | | | | | | | | incubation, adhesion to glass and polystyrene was 37 and 800 RLU/cm2, respectively. The relative adhesion coeffi- cient A for polystyrene was 62%, which was six times ) 2 0.2 3.0 1.1 1.1 1.5 1.7 1.2 6.5 4072.1 1.1 1.5 29.1 4.0 8.715 8.1 1.9 higher than that for the glass surface. Asaia cells are SD) on: ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ hydrophilic and attach to polymer surfaces with low surface ¡ Adhesion (RLU/cm

2.4 7.8 tensions (11, 21). The polar parts of the surface tension are 880 800 256 120 240 10.8 11.2 especially small, which may be explained by their long- chain molecular structures. The polystyrene surface is 4 8 7 3 7 6 3 7 5 level (mean

10 10 10 10 10 10 10 10 10 hydrophobic with low surface energy (40 mN/m at 20uC),

| | | | | | | | | and the glass surface is hydrophilic (70 mN/m at 20uC) (14). ) 3 A. bogorensis belongs to the group of acetic acid 1.1 3.1 1.9 3.0 2.0 1.4 1.7 8.9 7.6 bacteria that are tolerant to low pH levels and are able to ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ A. bogorensis 4 8 7 4 8 6 4 8 6 grow at pH 3.0 (8, 19, 26). Supplementation of the culture (CFU/cm 10 10 10 10 10 10 10 10 10

Planktonic cells medium with 10% cranberry juice resulted in reduction of | | | | | | | | | the pH from 3.5 to 2.5. However, A. bogorensis cells were

1.2 2.8 4.3 1.3 1.0 3.1 2.9 2.0 2.4 able to grow in this environment (1.1 | 107 CFU/ml) (Table 1). However, adhesion of bacterial cells in the

2 5 4 1 6 4 2 6 2 presence of cranberry juice was lower that that in the control

10 10 10 10 10 10 10 10 10 culture medium, especially on polystyrene. After 6 days of Glass | | | | | | | | | incubation, adhesion to the polystyrene surface was 256 ) 2 growth and adhesion abilities evaluated by plate count method and luminometry 2

1.6 2.0 1.7 6.2 1.2 4.1 1.7 1.7 3.6 RLU/cm , which was three times lower than the adhesion in

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ the control medium. We also calculated a lower value of 2 6 5 2 6 4 3 7 3

(CFU/cm relative adhesion coefficient A of 36%. 10 10 10 10 10 10 10 10 10 Adhered cells Culture media containing cranberry juice were formu- | | | | | | | | | lated with and without pH correction. After pH correction, the

Asaia bogorensis pH of the medium was reestablished at 3.5. Under these conditions, we observed growth of A. bogorensis cells (4.3 | )

2 8 15.64.6 1.4 2.3 13.27.6 2.0 7.7 18.06.5 1.5 5.8 0.1 2.1 1.0 3.3 0.3 2.8 10 CFU/ml). However, cell adhesion to polystyrene was ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ significantly lower than that in the control culture media Adhesion , vol/vol) on

(RLU/cm (Figs. 2 and 3). After 6 days of incubation in the culture % medium with cranberry juice after pH correction, adhesion to the polystyrene surface was 240 RLU/cm2, but the relative adhesion coefficient A was very small (4.41 ). We suspect 36 176 3 37.6 6 100 3 148 6 160 41.6 0 9.8 0 15.2 % day that this low A value may be a result of increased levels of Incubation planktonic bacterial cells and their reduced adhesiveness.

z Similar results were obtained by Lian and coworkers (15) for Staphylococcus aureus cells. Thus, the antiadhesive effect was not caused by the acidity of cranberries. % Many researchers have reported a correlation between 10 Effect of cranberry juice (10 various cranberry compounds and biofilm formation (2, 17, z cranberry juice 27), and these substances play an important role in reduction % 10 water cranberry juice with pH correction of the hydrophobicity of bacterial cells. Yang and coworkers

TABLE 1. Medium Flavored mineral waterFlavored mineral water 0Fruit-flavored mineral 1.4 (28) confirmed that cranberry juice components could 1190 ANTOLAK ET AL. J. Food Prot., Vol. 78, No. 6 impact bacterial cell initial adhesion by adhering to the uroepithelial-cell surfaces by proanthocyanidin extracts from cran- extracellular polymeric substances and impairing the berries. N. Engl. J. Med. 339:1085–1086. 11. Kregiel, D. 2013. Attachment of Asaia lannensis to materials adhesive capacity of the cells. commonly used in beverage industry. Food Control 32:537–542. Many studies on the antimicrobial and antiadhesive 12. Kregiel, D., A. Otlewska, and H. Antolak. 2014. Attachment of Asaia properties of cranberries have addressed action against bogorensis originating in fruit-flavored water to packaging materials. that are able to growth at neutral pH BioMed Res. Int. 2014:1–6. doi:10.1155/2014/514190. (e.g., E. coli, S. aureus, Staphylococcus epidermidis, and H. 13. Kregiel, D., A. Rygala, Z. Libudzisz, P. Walczak, and E. Oltuszak- Walczak. 2012. Asaia lannensis—the spoilage acetic acid bacteria pylori) (4, 15, 16, 23). However, the present study is the first isolated from strawberry-flavored bottled water in Poland. Food to describe the antiadhesive properties of cranberry juice Control 26:147–150. against acidophilic acetic acid bacteria, i.e., Asaia spp. 14. Lange, J., and Y. Wyser. 2003. Recent innovations in barrier A. bogorensis is often found as a contaminant of fruit- technologies for plastic packaging— a review. Packag. Technol. Sci. flavored mineral water. The growth of Asaia spp. in soft 16:149–158. 15. Lian, P. Y., T. Maseko, M. Rhee, and K. Ng. 2012. The antimicrobial drinks may cause flavor changes, package swelling, effects of cranberry against Staphylococcus aureus. Food Sci. ropiness, haze, or sediments (11–13). These bacteria are Technol. Int. 18:179–186.

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