Enhancing the Physicochemical Properties, Bioactivity, and Functional Applications of Fresh Jujube Juice Using Media Milling

Abstract

This study systematically evaluated the effects of media milling on the physicochemical properties, bioactive compound content, and functional applications of fresh jujube (Ziziphus jujuba Mill.) juice. Optimization experiments identified ideal conditions for nanoparticle production, including 5% solid content and a 180 min milling duration, resulting in significantly reduced particle sizes-volume-weighted average diameter (from 229.0 ± 1.0 to 25.0 ± 0.2 μm) and number-weighted average diameter (from 7.2 ± 0.0 to 0.1 ± 0.0 μm)-and improved dispersion stability. Media milling enhanced key physicochemical properties such as zeta potential, viscosity, and suspension stability, while also modifying color and pH. The process notably increased the content of bioactive compounds, including total flavonoids (from 2.9 ± 0.1 to 3.8 ± 0.0 mg catechin equivalent (CE)/g dry weight (DW)) and triterpenoids (from 15.4 ± 1.2 to 28.0 ± 4.9 mg oleanolic acid equivalent (OAE)/g DW). The antioxidant activity before and after media milling, assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, remained comparable. Fermentation with Lactiplantibacillus plantarum demonstrated that both blended and media-milled jujube juice can serve as effective substrates for substrate utilization and lactic acid production. Anti-inflammatory assays using RAW 264.7 macrophages revealed reduced nitric oxide production and lower levels of pro-inflammatory cytokines such as IL-1β, showcasing the juice's potential to modulate inflammation. In a dextran sodium sulfate (DSS)-induced colitis mouse model, media-milled jujube juice demonstrated safety, though it did not show significant protective effects. These findings position media-milled jujube juice as a promising functional food ingredient with potential applications in health promotion and disease management.

Figure 1

Optimization of nanoparticle preparation in fresh jujube juice using blending and media milling. Fresh jujube juice nanoparticles were prepared using a combination of blending and media milling, with a solid content of 5% in blended fresh jujube identified as the optimal concentration for achieving the smallest particle size and selected for further investigation, while a milling duration of 180 min was determined to be ideal for nanoparticle production. The figure illustrates (a) the experimental design and the effects of fresh jujube juice solid concentration and milling time on (b) volume-weighted average diameter, (c) number-weighted average diameter, (d) particle size distribution, (e) particle size span, (f) volume percentage, (g) polymer dispersity index (PDI), (h) number percentage, and (i) D90 volume-weighted mean diameter and reduction ratio. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons tests, comparing different groups at each time point for (b) and (c), between groups for (d) and (e), and across time points for (g) and (h). Different letters indicate significant differences (p < 0.05) among groups. Data are presented as mean ± SD.

Figure 2

Effect of media milling on fresh jujube juice nanoparticles: appearance, microstructure, color, physicochemical properties, and stability. (a) The visual appearance of fresh jujube fruits (scale bar: 1 cm) and a microscopic image showing the cellular structure (scale bar: 50 μm), (b) optical microscopy images of jujube juice before and after media milling, illustrating particle size reduction, (c) scanning electron microscopy (SEM) images highlighting the changes in microstructure after media milling, (d) photographs of jujube juice at different milling times (0–180 min), showing color and dispersion changes, (e) color space variables (L*, a*, b*, and ΔE) of jujube juice samples as a function of milling time, indicating color changes with increased milling duration, (f) pH values of jujube juice samples at various milling times, showing a gradual increase over time, (g) zeta potential measurements demonstrating the effect of milling time on the surface charge of nanoparticles, (h) viscosity of jujube juice as a function of milling time, showing an increase with longer milling durations, (i) backscattering (BS) ratio profiles during 24-h storage, comparing stability across milling times, (j) aggregation percentage of jujube juice samples during 24-h storage, indicating improved stability with increased milling time. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons tests, comparing the changes across time points for (e), (f), (g), and (h). Abbreviations: Blended fresh jujube (F–B); media-milled fresh jujube for 180 min (F-M180). Different letters indicate significant differences (p < 0.05) among groups. Data are presented as mean ± SD.

Figure 3

Effect of media milling on total polyphenol, flavonoid, triterpenoid content, and antioxidant activity of fresh jujube juice nanoparticles. (a) the total polyphenol content of jujube juice across different milling times (0–180 min), (b) total flavonoid content, (c) total triterpenoid content, (d) total bioactive compound content, including polyphenols, flavonoids, and triterpenoids, (e) individual triterpenoid components (betulinic acid, oleanolic acid, and ursolic acid) quantified in blended (F–B) and 180 min media-milled jujube juice (F-M180). (f) DPPH inhibition as a measure of antioxidant activity for F–B and F-M180, with IC50 values. (g) FRAP inhibition for F–B and F-M180. (h) ABTS inhibition for F–B and F-M180. Statistical analyses included one-way ANOVA with Tukey’s multiple comparison tests for panels (a), (b), and (c), and two-tailed Student’s t tests for (e). Dose–response curves (panels f–h) were analyzed using Log(inhibitor) vs normalized response – Variable slope models. Abbreviations: F–B, blended fresh jujube; F-M180, media-milled fresh jujube for 180 min. Different letters indicate significant differences (p < 0.05) among groups. Data are presented as mean ± SD.

Figure 4

Utilization of blended fresh jujube (F–B) and media-milled fresh jujube for 180 min (F-M180) as substrates by Lactiplantibacillus plantarum for lactic acid production. (a) Schematic of the fermentation process: F–B and F-M180 were pasteurized and inoculated with L. plantarum. Changes in sugar content and glucose utilization were monitored to assess lactic acid production, (b) sugar content, (c) glucose concentration, (d) pH changes, (e) titratable acidity. Fermentation was carried out over 48 h. Statistical analyses were performed, with significant differences indicated by different letters (within-group comparisons at different time points) or asterisks (F–B vs F-M180; *p < 0.05, **p < 0.01, ****p < 0.0001). The results show efficient substrate utilization and acid production, with F–B and F-M180 showing distinct fermentation dynamics over time. Data are presented as mean ± SD.

Figure 5

Effect of blended fresh jujube (F–B) and media-milled fresh jujube for 180 min (F-M) on RAW 264.7 macrophage cells stimulated by lipopolysaccharide (LPS). (a) Experimental design illustrating the treatments and key measurements, including cell viability, NO production, and cytokine levels (IL-1β, IL-6, TNF-α), (b) Cell viability, (c) nitrite (NO) production, (d) IL-1β secretion, (e) IL-6 secretion, and (f) TNF-α secretion. RAW 264.7 cells were treated with F–B, F-M, and ursolic acid for 24 h. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Different letters indicate significant differences (p < 0.05) among groups. Data are presented as mean ± SD.

Figure 6

The impact of blended fresh jujube (F–B), media-milled fresh jujube for 180 min (F-M), and the supernatant of F-M (F–S) on body weight changes and phenotype in a DSS-induced colitis mouse model. (a) Schematic diagram of the experimental design, (b) body weight changes during the experimental period, (c) body weight on day 18, (d) colon weight. (e) colon length, and (f) colon weight normalized to body weight. Five-week-old male BALB/c mice were acclimatized for 10 days before receiving oral gavage with F–B, F–M, or F–S (0.1 mL/mouse/day) for 18 days. From day 18 to day 24, 2% DSS (dextran sulfate sodium) was added to the drinking water to induce colitis, followed by regular water until the mice were sacrificed on day 28. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s multiple comparisons tests. Groups sharing the same letter are not significantly different (p > 0.05). Data are presented as mean ± SD.