Chemical reagents
Sodium hydroxide (NaOH), ethyl acetate, ethanol (96%), and methanol were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2′-Azino-bis(3-.
ethylbenzothiazoline-6-sulfonic acid) diammonium salt, potassium persulfate, Trolox (6-hydroxy- 2,5,7,8-.
tetramethylchroman-2-carboxylic acid), Folin-Ciocalteu reagent, sodium carbonate, gallic acid, catalase from bovine liver, and phenolic and organic acid standards were purchased from Sigma‒Aldrich (Merck, Darmstadt, Germany). Acetonitrile for HPLC was purchased from Honeywell (USA), whereas phenolphthalein and hydrochloric acid (35–38%) were purchased from Chempur (Piekary Śląskie, Poland).
Berry juices
Four types of commercially available NFC (not from concentrate) berry juices — elderberry (Sambucus nigra), chokeberry (Aronia melanocarpa), blackcurrant (Ribes nigrum), and cranberry (Vaccinium macrocarpon) — were sourced from Batom (Kraków, Poland) for this study. Juices from three distinct production batches were used to ensure variability and representativeness in the analysis. According to the manufacturer’s declaration, the juices were cold-pressed from whole fruits and subjected to mild continuous flow pasteurization, ensuring that the temperature did not exceed 85 °C, with no additional preservation techniques or agents applied. To confirm that the juices would not introduce additional microorganisms, a microbial analysis was conducted. Overall, the results demonstrated the absence of mesophilic, psychrophilic, Enterobacteriaceae, and lactic- and spore-forming bacteria in the juice microflora, ruling out juices as potential sources of contamination.
SPE fractionation of berry juices
Two milliliters of a berry juice sample were partitioned into two fractions, namely, phenolic and anthocyanin fractions, via solid-phase extraction (SPE) with C-18 columns, following the procedure outlined by19. After drying and reconstitution, qualitative and quantitative analyses were conducted on the fractions as detailed in section UHPLC-DAD and UHPLC-ESI-HRMS/MS analysis of phenolic compounds. Prior to the antibacterial assessments, the dried fractions were redissolved in tryptic soy broth (TSB medium).
Survival of B. cereus in the presence of berry juices and their individual components
The study utilized two bacterial strains: Bacillus cereus AK1, a newly isolated strain from cold-brewed green tea (accession number OQ875857 in the GenBank National Centre for Biotechnology Information database), and Bacillus cereus ŁOCK 0807, obtained from the Pure Culture of Industrial Microorganisms of the Institute of Fermentation Technology and Microbiology ŁOCK 105 (Łódź, Poland). Prior to the experiments, the strains stored in cryovials at -80 °C were activated through two successive subcultures in TSB medium (Merck, Darmstadt, Germany; pH 7.4 ± 0.1). Bacterial growth was monitored in media supplemented with a single type of juice at final concentrations of 5%, 7.5%, or 10% (v/v), either in its native (acidic) or neutralized form. These concentrations mimic typical commercial ready-to-drink beverages. In this context, higher supplementation levels had been found to be detrimental from a sensory perspective. For the neutralized juices, the pH was adjusted to 7.4 (± 0.1) using a 1 M NaOH solution. With respect to the fractional components of individual berry juices —phenolics and anthocyanins — the amounts used in the experiments were 3 and 5 milligrams per milliliter of the sample solution, respectively. The prepared solutions were inoculated with a 1% (v/v) 24-hour liquid inoculum containing approximately 106 bacterial colony-forming units per milliliter (CFU/mL) and then incubated at 30 °C for 24 h. For comparison purposes, positive controls were inoculated with TSB without juices or their fractions. The viable counts of individual B. cereus strains were evaluated at specific time intervals during the incubation period via the plate method. This included measurements at 0, 1, 6, 10, and 24 h for the juice supplements and at 0 and 24 h for the fractional components. The toxicity of growth-inhibitory juice supplements toward B. cereus cells was also assessed after 24 h of treatment in the absence and presence of 500–10,000 U of catalase. After each incubation period, the cultures were diluted and spread on tryptic soy agar (TSA) plates and then incubated for 24 h at 30 °C. The viable counts were expressed in CFU/mL and alternatively calculated as a percentage of the control.
pH and acidity of the juices
The pH of the examined juices was determined via a pH meter CP-411 (Elmetron, Zabrze, Poland). The titrable acidity (TA) analysis was performed using a 0.1 M solution of sodium hydroxide and a 1% solution of phenolphthalein. The TTA results were expressed as the volume (mL) of 0.1 M NaOH required to titrate one milliliter of a juice sample (mL 0.1 M NaOH/mL).
Antioxidant capacity of the juices
The antioxidant capacity (AC) of the juices was determined via the DPPH and ABTS methods, following the protocols described by20,21. The reduction of DPPH• radicals was measured at a wavelength of 517 nm, while the reduction of ABTS•+ radicals was measured at a wavelength of 734 nm via a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DPPH and ABTS antiradical activities of the juices were calculated using standard curves with Trolox as the reference compound and expressed as milligrams of Trolox equivalents per liter of the juice sample (mg TE/L).
Total phenolic content of the juices via spectrophotometry
The total phenolic content (TPcF−C) of the berry juices via spectrophotometry was determined following the Folin‒Ciocalteu (F‒C) method as described by22. The analysis was conducted via a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 96-well plates. The TPcF−C in the samples was calculated using a standard curve for gallic acid and expressed as milligrams of gallic acid equivalents per liter of the juice sample (mg GAE/L).
UHPLC-DAD and UHPLC-ESI-HRMS/MS analysis of phenolic compounds
UHPLC-DAD and UHPLC-ESI-HRMS/MS analyses of phenolic compounds in the juice samples were performed via a UHPLC + Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diode array detector and a Transcend™ TLX-2 multiplexed LC system coupled to a Q-Exactive hybrid quadrupole‒orbitrap mass spectrometer with a heated electrospray ionization (HESI-II) source (Thermo Scientific, Hudson, NH, USA) according to23, with some modifications. Juice samples were diluted in mobile phase at a ratio of 1:10 (v/v) and filtered through a nylon membrane filter (0.22 μm pore size) prior to analysis. The chromatograms were recorded at 270 nm for flavan-3-ols and hydroxybenzoic acids, at 320 nm for hydroxycinnamic acids, and at 520 nm for anthocyanins. The identification of phenolic compounds was performed by matching their retention times, spectral characteristics, full-scan mass spectra in negative and positive ionization modes, and MS/MS fragmentation patterns with those of pure standards analyzed under identical conditions. The quantification of individual phenolic compounds was carried out using the external standard method. The results were expressed as milligrams of phenolic compounds per hundred milliliters of the juice sample (mg/100 mL). The concentration ranges, correlation coefficients (R2), limits of detection (LODs), and limits of quantification (LOQs) for the phenolic compounds investigated are given in Table S1.
HPLC‒DAD analysis of organic acids
HPLC-DAD analysis of organic acids in the juice samples was performed via a UHPLC + Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diode array detector according to24, with some modifications. Juice samples were diluted in mobile phase at a ratio of 1:10 (v/v) and filtered through a nylon membrane filter (0.45 μm pore size) prior to analysis. Chromatographic separation of the phenolic compounds was achieved using a A11606 C18 column (2.1 × 150 mm, particle size 2.6 μm; ATC, Waltham, MA, USA). The column was maintained at 30 °C, and the flow rate was 0.8 mL/min. The mobile phase solutions were 20 mM NaH2PO4 buffer, pH 2.3 (A), and acetonitrile (B). The following gradient program was used: 0 min, 0% B; 0–5.5 min, from 0 to 80% B; 5.5–10.5 min, constant at 80% B; 10.5–19.5 min, from 80 to 0% B; 19.5–25 min, constant at 0% B. Chromatograms were recorded at 210 nm. The identification of organic acids was based on a comparison of the retention times of analytes with those of available standards (oxalic acid, tartaric acid, malic acid, ascorbic acid – AsA, citric acid – CA, and fumaric acid). The external standard method was used to determine the concentrations of individual organic acids. The results were expressed as milligrams of organic acids per hundred milliliters of the juice sample (mg/100 mL).
Reproducibility
All assays were performed on three independently produced replicates of each berry juice, and the data are expressed as means ± standard deviations. Figures were created via GraphPad Prism (version 9.00, GraphPad Software Inc., San Diego, CA) and Excel (Microsoft 365, USA). Statistical analyses were performed via R software (version 4.2.2; PBC, Boston, MA). The normality of the distribution was assessed with the Shapiro‒Wilk test, and variance homogeneity was evaluated using Bartlett’s test. Differences among the examined parameters were analyzed using one-way ANOVA, followed by Tukey’s honest significance test (HSD) and Duncan’s test (MRT), with a significance level of α = 0.05. The Kruskal‒Wallis test was used to evaluate differences between juice-treated and untreated bacterial cells (assessments involving catalase and fractional components of the juices).
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- Source: https://www.nature.com/articles/s41598-024-79155-y