The reagents used in the study were kuromanin chloride, ≥ 96.0% purity (Extrasynthese, France), formic acid, ≥ 98.0% purity (Sigma-Aldrich, United States), potassium chloride, 99.0–100.5% purity (Sigma-Aldrich, United States), sodium acetate, ≥ 99.0% purity (Sigma-Aldrich, United States), ethanol, ≥ 96.0% purity (Sigma-Aldrich, United States), methanol, ≥ 99.0% purity (Sigma-Aldrich, United States), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ≥ 97.0% purity (Sigma-Aldrich, United States), L-ascorbic acid, 99.7–100.5% purity (Merck KGaA, Germany), isopropyl alcohol, ≥ 99.5% purity (Honeywell, United States), Folin–Ciocalteu reagent, 2 M (Sigma-Aldrich, United States), sodium carbonate, ≥ 98% purity (Sigma-Aldrich, United States), gallic acid, ≥ 99.0% purity (Sigma-Aldrich, United States), 2,2-diphenyl-1-picrylhydrazyl (DPPH), (Sigma-Aldrich, United States), hydrochloric acid, ≥ 37.0% purity (Sigma-Aldrich, United States), phosphoric acid, ≥ 85.0% purity (Chem-Lab, Belgium).

Black chokeberry (Aronia melanocarpa (Michx.) Elliott), highbush blueberry (Vaccinium corymbosum L.), bilberry (Vaccinium myrtillus L.), honeysuckle berry (Lonicera caerulea L.), elderberry (Sambucus nigra L.), black currant (Ribes nigrum L.), red currant (Ribes rubrum L.), bog cranberry (Vaccinium oxycoccos L.), strawberry (Fragaria × ananassa Duchesne ex Rozier), and raspberry (Rubus idaeus L.) pomaces were provided by the local juice producer LLC “Very Berry” (Dārzciems, Gaujiena parish, Smiltene county, Latvia) in February 2025. Berry press residues were stored in polyethylene bags at −20 °C.

To determine the berry press residue anthocyanin profile, the European Pharmacopoeia 7.0 instrumental method “Fresh bilberry fruit dry extract, refined and standardised” (01/2016:2394) with some modifications was used. Before analysis, the berry press residue extracts were filtered through 0.45 μm membrane filters. Chromatography vials were filled with 1.00 mL sample and determination of anthocyanin profile was carried out using Waters Acquity UPLC H- class (Waters, United States) consisting of a ultra-high-pressure gradient unit (Quaternary Solvent Manager), an auto injector (FTN), a column oven (CH-A), a photo-diode array PDA eλ detector and MS detector (XEVO TQD with Zspray ESI/APSI/ESCi) with electrospray ionisation (ES-), capillary voltage 3.50 kV, cone voltage 40 V, ion source temperature 150 °C, spray temperature 400 °C, spray gas flow 300 L/h, a cone gas counterflow of 50 L/h, a collision energy of 15 V, argon at 3,5 ∙10⁻³ mBar as collision gas, an ion recording range of 200–1700 m/z (0.15 s), and an operating mode of SIR. Anthocyanin separation was carried out at 35 °C using an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 150 mm). The mobile phases were deionised water (A), methanol (B), formic acid (C) and acetonitrile (D) (Table 1). The flow rate was set to 0.250 mL/min, and the gradient elution was set according to Table 1. Data was collected using Waters Empower software. Identification of individual anthocyanins was based on the PDA detection at 520 nm and further UPLC-ESI-MS/MS analysis to identify the molecular ions ([M]+) and diagnostic fragment ions corresponding to the anthocyanin aglycones. MS/MS spectra were further compared with reference spectra available in the mzCloud mass spectral database (Thermo Fisher Scientific), data reported in the European Pharmacopoeia and relevant literature to support compound assignment.

Quantification of individual anthocyanins was done using external calibration prepared from Cyanidin-3-glucoside (kuromanin chloride) ≥ 96.0% (Extrasynthese, France). Calibration curve was prepare din the concentration range from 0.01 mg cyanidin-3-glucoside (C3G) eq./mL to 2.4 C3G eq./mL (Supplementary Figure 3).

Data visualisation and statistical analysis have been done using the programme “R” (version 2023.06.2 + 561), using ggplot2 (version 3.5.1), corrplot (version 0.95), dplyr (version 1.1.4), GGally (version 2.3.0) and readxl (version 1.4.3) data packages. Correlation analysis between TAC, TPC, and DPPH was performed using Spearman’s rank correlation coefficients to evaluate the associations between total anthocyanin content, total polyphenolics, and antioxidant activity. Individual correlations between drying methods in each berry press residue sample, depending on the temperature, were done using Pearson’s correlation analysis for TAC, TPC and DPPH. A pairwise correlation matrix was constructed using the ggpairs function with LOESS regression fits for identification of non-linear trends and kernel density estimates.

The effect of conventional and vacuum drying at different temperatures on total polyphenolic content (TPC) in 10 types of berry press residues was evaluated using the Folin–Ciocalteu method. Figure 1 shows the changes in total polyphenol concentration depending on drying temperature and method for each berry species (Figure 1).

Freeze drying, used as a baseline control to indicate possibly the lowest degradation within the samples, yielded the highest polyphenolic retention across all tested berry species, with mean TPC values as high as 8,377 mg/100 g DW of chokeberry press residues.

The concentration of polyphenols decreased in the samples with increasing temperature from 30 °C to 90 °C. Exceptions in this trend were observed for cranberries (using both drying methods) and strawberries (using vacuum drying). These findings confirm the widely reported efficiency of freeze drying in preserving thermolabile bioactive compounds due to the absence of oxidation and thermal stress (Ma et al., 2023). Freeze drying thus represents a method that can induce minimal polyphenolic degradation, providing polyphenolic retention similar to fresh material.

Conventional hot air drying showed relatively stable TPC values in the temperature range from 30 °C to 75 °C, indicating that moderate drying temperatures do not necessarily result in polyphenolic loss and, in some cases, are comparable to the used control freeze drying. However, at 90 °C, the TPC decreased sharply and was reduced significantly in most samples (Figure 1). It was observed that the drying temperature and TPC showed a negative relation, where increasing the temperature significantly reduced the TPC. The sharp reduction at 90 °C suggests a temperature threshold at which the degradation of polyphenolics is likely accelerated, possibly due to the combined effects of thermal breakdown and oxidation. The non-linear response of TPC to temperature implies that polyphenolic stability cannot be assumed to decline gradually, but rather that certain processing conditions can trigger increased degradation.

Vacuum drying displayed a specific pattern where the characteristics of conventional and freeze drying were combined. At lower temperatures (30–60 °C), mean TPC values between vacuum and freeze drying were statistically indistinguishable (Figure 1). At 75 °C, vacuum drying shows higher TPC retention than conventional drying, suggesting that reduced oxygen availability delays degradation. However, at 90 °C, TPC were reduced significantly for most of the tested berries, closely resembling the values of conventional drying at the same temperature. These findings suggest that oxygen exclusion is effective at mitigating degradation under mild to moderate temperature conditions, but once the temperature exceeds certain thresholds, the exclusion of oxygen is insufficient to mitigate the degradation effects.

When comparing phenolic contents between samples dried at 30 °C and 90 °C, clear berry- and method-specific differences were observed (Figure 2). Generally, the TPC values decreased significantly with the temperature increase for all the berries and methods evaluated; however, the magnitude at which the losses happened varied. Chokeberry and honeysuckle berry TPC decreased by 50% or more under conventional and vacuum drying, indicating high susceptibility of these matrices to thermal degradation. In contrast, raspberries, strawberries, and cranberries exhibited a more moderate decline in TPC (2–19%), suggesting greater thermal stability (Figure 2).

Across most berry types, vacuum drying consistently showed less degradation than conventional drying, leading to smaller relative differences between the low and high drying temperatures. This protective effect was particularly evident in raspberry, strawberry, and cranberry samples, where vacuum drying resulted in only 2–5% TPC loss (Figure 2). A strong trend was observed: drying at 90 °C resulted in lower TPC than 30 °C, confirming that the temperature is the dominant driver of phenolic compound degradation, regardless of the drying method used.

All drying methods showed high TPC variability, depending on the berry type and matrix composition. Berries with more robust cell structure (e.g., chokeberry) or berries with naturally higher TPC may withstand processing better than more delicate species such as strawberry. As the results indicate, it is important to assess the possible matrix effects across different species since the drying conditions cannot be generalised without considering inherent compositional differences. Taken together, the obtained results highlight a significant interaction between drying method and temperature regarding TPC. Freeze drying provides a reliable retention of phenolic compounds followed by vacuum drying at moderate temperatures (30–60 °C), giving similar TPC retention with lower energy requirement and processing time than freeze drying. However, both conventional and vacuum drying exhibit a degradation threshold between 75 °C and 90 °C, beyond which phenolic compounds were rapidly degraded, emphasising the need for controlled conditions during the drying process as well as balancing the energy consumption with final product quality.

The total anthocyanin content (TAC) of the different berries was strongly influenced by both drying methods and temperature. The highest TAC was observed in chokeberry dried under vacuum at 45 °C, reaching up to 3,826 mg/100 g DW, while the lowest value was recorded in raspberry subjected to conventional drying at 90 °C (26 mg/100 g DW). These radically different values represent and highlight the combined effects of berry type and drying conditions on anthocyanin stability.

In general, chokeberry exhibited the highest TAC across all drying temperatures and methods, confirming its status as one of the richest natural sources of anthocyanins. Also, blueberry, bilberry, and honeysuckle berry maintained relatively high TAC values. In contrast, cranberry and raspberry showed considerably lower anthocyanin levels and distinct degradation patterns across the drying temperatures using both drying methods (Figure 3). The control treatment – freeze drying – generally preserved anthocyanins more effectively than the thermal drying methods, with values close to or exceeding those obtained at the lowest (30 °C) conventional or vacuum drying temperatures. Vacuum drying tended to provide higher TAC values than conventional drying, particularly in the temperature range 30–60 °C, where anthocyanin retention remained relatively high compared to the decrease between 75 and 90 °C. At 90 °C, anthocyanin degradation was evident in all tested berry samples, although the extent of reduction was species dependent. Anthocyanin degradation for cranberry, blueberry, elderberry, honeysuckle berry, raspberry, and strawberry showed a clear linear relation (Figure 3).

Reduction in TAC was observed when comparing berry press residues dried at 30 °C and 90 °C (Figure 4). Significant reduction was observed across all berry types investigated, with the highest reduction observed in raspberries dried conventionally (up to 59.4%). Least reduction in TAC was observed for conventionally dried redcurrant press residues (9.7%). Regarding TAC content and the drying method used, it was observed that the vacuum drying method, in all types of berry press residues, retained more anthocyanins than the conventional drying (Figure 4). This indicates that the specific degradation of anthocyanins is dependent on the presence of oxygen – prolonged exposure in elevated temperatures with oxygen present (conventional drying) leads to more pronounced anthocyanin degradation. In berries like bilberry, blueberry, chokeberry, where the TAC contents are the highest, the degradation using vacuum drying was recorded to be from 20 to 26% as opposed to the >40–50% in conventional drying (Figure 4).

The results demonstrate the heat sensitivity of anthocyanins in berry press residue and highlight the importance of selecting the appropriate drying method for the preparation of dried products. Variation in TAC among berry species highlights the importance of matrix composition in stabilising or protecting anthocyanins: bilberry and blueberry showed good anthocyanin retention, while raspberry and honeysuckle berry anthocyanins were the most heat sensitive. Moreover, for retention of anthocyanins, it is advisable to choose drying methods that avoid the presence of oxygen and use temperatures in the range from 30 °C to 60 °C.

DPPH radical scavenging activity was determined for all the berry press residue samples dried at different temperatures with each of the tested methods. Freeze drying, the mildest of the drying methods that avoids elevated temperatures and oxygen exposure, showed the highest DPPH radical scavenging activity for all of the tested berries, with the highest activity observed for freeze-dried chokeberry press residues (12,930 mg TE/100 g DW), followed by honeysuckle berry (8,014 mg TE/100 g DW). These results indicate that maintaining low processing temperatures is critical for preserving antiradical scavenging activity, as significant reductions were observed for both thermal drying methods even at 30 °C. Vacuum drying at moderate temperatures (30–60 °C) was proven to retain the antioxidants responsible for DPPH activity better than conventional drying. While most berry press residues followed this pattern, some berry-specific deviations were evident, for example, honeysuckle berry showed increasing DPPH radical scavenging activity over the tested range of temperatures (Figure 5). This suggests that, in addition to polyphenolics, other compounds (e.g., Maillard reaction products formed during thermal processing) may contribute to the antioxidant potential. These results are in line with the hypothesis that both temperature and the drying method can affect the antioxidant capacity of a product during processing. Although the trend of DPPH radical scavenging activity is non-linear with the TPC or TAC, it is worth noting that vacuum drying is the optimal method to be used for retention of valuable antioxidants in this type of waste biomass.

The relationship between total polyphenolic content (TPC), total anthocyanin content (TAC), and antioxidant activity (DPPH) was assessed across all drying treatments, and the results are summarised in Figure 6. Strong positive associations were observed between TPC and DPPH (r = 0.891^***), while TAC correlated positively with both TPC (r = 0.765^***) and DPPH (r = 0.662^***). These results suggest that total polyphenolic levels were the dominant contributors to antioxidant potential, whereas anthocyanins, while important, were not the sole determinants of radical-scavenging capacity. Importantly, these correlations were consistent across different drying methods, highlighting that the strength of association was not simply an artefact of processing. Within conventional, freeze-, and vacuum-dried samples, TPC remained strongly correlated to DPPH (r = 0.895, 0.964, and 0.901^***), emphasising the robustness of this relationship. TAC–TPC correlations were also significant under all conditions (r = 0.718–0.891), with the closest coupling observed for freeze-dried material, suggesting that anthocyanins behave as a relatively stable sub-fraction of total phenolics in this treatment, especially under milder processing conditions. By contrast, TAC–DPPH associations were slightly weaker (r = 0.627–0.782), pointing toward a more indirect or variable contribution of anthocyanins to antioxidant capacity, however, statistically, they show a strong relation.

The smoothed regression fits (Figure 6) further emphasise these relationships - the TPC–DPPH relation trend showed a nearly linear fit across the observed range, with a tendency toward stronger activity at higher phenolic concentrations, whereas TAC displayed greater dispersion and wider confidence intervals, particularly at intermediate values. Density plots along the diagonal (Figure 6) highlight that freeze-dried samples exhibited a broader distribution of both TPC and DPPH values, consistent with their higher correlation strength, while vacuum- and conventionally dried samples clustered more tightly. These findings suggest that while anthocyanins contribute to antioxidant activity, the potential of total phenolics is a more important and more consistent contributor. Thus, phenolic content as a whole, rather than anthocyanin concentration alone, appears to drive the radical-scavenging properties of the studied material, with processing method influencing the magnitude of the effect but not the overall pattern of association.

Analysis of individual anthocyanin profiles revealed that certain structures were markedly more sensitive to thermal degradation than others (Table 2). Across all berries and drying methods, 24 individual anthocyanins were found. Cyanidin 3-O-sambubioside showed the highest mean reduction (54.2%), indicating exceptional thermal lability. Similarly, cyanidin 3,5-O-diglucoside and cyanidin 3-O-sophoroside were highly degraded (41.9 and 35.1% on average, respectively), with only marginal differences between conventional (C) and vacuum (V) drying (Table 2). By contrast, rutinoside derivatives such as cyanidin 3-O-rutinoside displayed much lower reductions (19.8% under C, 16.0% under V), suggesting greater structural resilience to both heat and oxygen exposure. A compilation of chromatograms of the analysed blackcurrant shows the reduction in differently dried berry press residue anthocyanin profiles (Figure 7).

A consistent pattern emerged in which rutinosylated and glucosylated forms (e.g., cyanidin 3-O-rutinoside, delphinidin 3-O-glucoside, malvidin 3-O-glucoside) were comparatively stable, typically showing 18–30% reduction, whereas diglucosides and sambubiosides were markedly less stable, with 40–60% losses (Table 2). This structural trend suggests that acylation or rutinosylation may confer protection against heat-driven cleavage and oxidative breakdown, whereas more labile linkages (e.g., sophoroside and sambubioside groups) are especially vulnerable.

When comparing drying methods, vacuum drying systematically was able to avoid anthocyanin loss across almost all analysed anthocyanins and samples, although the magnitude varied. For instance, cyanidin 3-O-arabinoside decreased by 38.5% under conventional drying but only 25.1% under vacuum, while delphinidin 3-O-galactoside dropped from 37.0% (C) to 23.9% (V) (Table 2). However, for compounds already highly unstable (e.g., cyanidin 3-O-sambubioside), the protective effect of vacuum was limited, with degradation remaining high under both drying methods (59.1% vs. 49.3%) (Table 2).

Together, these findings highlight that anthocyanin degradation is not uniform across berry matrices but strongly influenced by the molecular structure of individual anthocyanins. Glycosylation pattern appears to be a major determinant of thermal stability, with rutinosides and simple glucosides showing the greatest resilience, while sambubiosides and diglucosides are disproportionately degraded. These trends provide novel insight into structure–stability relationships of anthocyanins during drying, underscoring the importance of considering individual anthocyanin chemistry, in addition to total anthocyanin content, when evaluating the nutritional and functional quality of processed berry products.