Goldenberry (Physalis peruviana L.) juice inhibits foodborne pathogens while promoting probiotic bacterial growth

Abstract

The antibacterial properties of goldenberry (Physalis peruviana L.) juice were investigated against foodborne pathogens, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes. At different time intervals (0, 24, 48, and 72 h), the growth inhibition of these pathogens was assessed in broths, skim milk, and whole milk supplemented with 10% goldenberry juice. The effects of juice on the development of L. plantarum, B. lactis Bb12, B. angulatum DSMZ 20,098, L. rhamnosus TISTR 54, and L. acidophilus 20,552 were investigated. Goldenberry juice reduced the growth of L. monocytogenes, S. aureus, P. aeruginosa, and E. coli by 1–3 log CFU/mL in both broth and milk. The strength of inhibition varied by bacterial type, with S. aureus and L. monocytogenes showing greater sensitivity than the Gram-negative bacteria. The juice, when added to both MRS broth and milk, increased the growth of probiotic and lactic acid bacterial strains (1–4 log CFU/mL). The data showed that goldenberry extract exhibits antimicrobial and growth-inhibitory effects, which are retained and enhanced when applied in dairy systems, making it a promising natural preservative for improving the microbial safety of milk-based products.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-57257-z.

Introduction

The leading cause of death worldwide is infectious diseases, caused by microbes such as bacteria, viruses, fungi, and parasites that enter the body and multiply¹. Bacteria are the primary cause of most illnesses, and symptoms vary depending on the affected body part. Skin infections are caused by the harmful bacteria Streptococcus and Staphylococcus, which can cause infections or serious illnesses². Salmonella, E. coli, Listeria monocytogenes, and Vibrio have all been connected to food-borne illness^3,4. Additionally, according to Flores-Mireles et al.⁵, Enterococcus faecalis, Klebsiella, Proteus, and uropathogenic E. coli (UPEC) are the primary causes of urinary tract infections. Various pathogenic bacteria employ similar tactics to spread infection and sickness⁶. Antibiotics are commonly used to treat most bacterial illnesses. Over the past three decades, more research has been conducted on natural chemicals, increasing the body of evidence supporting their antibacterial action^7–9. Furthermore, it has been demonstrated that thousands of chemicals isolated from plants have therapeutic or antimicrobial qualities¹⁰. Salmonella enterica, Listeria monocytogenes, and enterohemorrhagic E. coli O157:H7 are the top three bacterial pathogens responsible for foodborne infections, according to Scallan et al.¹¹ and Yang et al.¹². Although probiotics -specifically lactic acid bacteria, such as L. casei, L. plantarum, and L. rhamnosus- are widely used in the food industry for fermentation, medical professionals are increasingly interested in them due to their potential health benefits. Probiotic therapy is now considered an effective way to improve gut health, rather than antibiotics. Studies have shown that probiotic strains can effectively treat acute diarrhea and prevent inflammatory bowel disorders^5,13,14. Additionally, for the treatment of several illnesses, natural antimicrobials and prebiotics offer attractive alternatives to synthetic chemical antibiotics. Nohynek¹⁵; Saarela et al.¹⁶; Bayas-Morejon et al.¹⁷ claim that they are rich in bioactive compounds that combat bacterial illnesses and promote the growth of probiotic species. Numerous studies^4,18 report that blueberry juice can limit the growth of foodborne bacterial pathogens but does not significantly reduce the growth of probiotic bacteria. Blackberries are rich in antioxidants and other nutrients, including fiber, ellagic acid, salicylic acid, proanthocyanidins, anthocyanins, and other flavonoids^19–23. Several of these compounds have been shown to possess antibacterial and anti-cancer effects^14,24.

Numerous fruits have been demonstrated to offer protection against heart disease and other ailments²⁵; cancer, inflammation, aging, viral infections, and Alzheimer’s disease^26,27; skin cancer, allergies, and cutaneous damage^26,28. Examples of these fruits include grapes, berries, pomegranates, and apples. Their high antioxidant activity accounts for most of their health benefits²⁹.

Thus far, several phytochemicals possessing diverse modes of action have been recognized as antimicrobial agents. The most extensive research has focused on the ability of berry extracts to inhibit harmful germs. Condensed tannins, or proanthocyanidins, are found in cranberries and blueberries in particular. These tannins can prevent symptomatic urinary tract infections (UTIs), which are nearly always caused by pathogenic strains of E. coli^30,31. E. coli, a common cause of diarrhea and extra-intestinal infections, has been demonstrated to be effectively combated by phenolic compounds found in berries, which are consumed in large quantities throughout Europe³². Furthermore, phenolic compounds are beneficial against Salmonella and Campylobacter intestinal infections³³. Secondary metabolites are fruit-derived antimicrobial compounds that function by suppressing infections and preventing chronic illnesses. According to Ramadan and Moersel³⁴, who produced goldenberry juice, the results were deemed excellent, and the juice has the potential to be highly valued compared to other generally accessible items on the market³⁵.

Natural plant extracts are widely used as flavoring agents in dairy products due to their sensory and functional properties^4,36. One potential application for blackberry and goldenberry juice is as flavor enhancers. Additionally, it is a naturally occurring antibacterial substance^37,38. Golden berries may develop into a commercial fruit of special importance to upmarket eateries and bakers worldwide^39,40. This was the approach that created kiwifruit markets in the 1960s and resulted in an annual crop worth millions of dollars. Recent advances in biological and agricultural research have highlighted the growing importance of integrating modern analytical and computational approaches to improve the accuracy of biological evaluations and the interpretation of complex experimental data. Advanced statistical and predictive methods have been increasingly applied in agricultural and food-related studies to support early detection, classification, and optimization processes, thereby enhancing the reliability and applicability of research outcomes. Such approaches may also improve the interpretation of microbial responses and functional food investigations involving natural bioactive compounds⁴¹.

Despite the growing interest in fruit-derived bioactive compounds as natural antimicrobial agents, limited studies have investigated their selective activity against pathogenic bacteria while simultaneously supporting the growth of beneficial probiotic microorganisms. Therefore, the present study aimed to evaluate the dual-action potential of thermally processed goldenberry juice as a natural functional ingredient capable of inhibiting food-borne pathogens while promoting probiotic bacteria. It was hypothesized that goldenberry juice possesses selective antimicrobial activity against pathogenic bacteria, including E. coli, P. aeruginosa, S. aureus, and L. monocytogenes, while enhancing the growth of beneficial strains such as L. plantarum, B. lactis Bb12, B. angulatum DSMZ 20,098, L. rhamnosus TISTR 54, and L. acidophilus 20,552. The findings may support the use of goldenberry juice as a natural antimicrobial and functional food ingredient to improve food safety and probiotic viability.

Materials and methods

Microorganisms

Food-borne strains

Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and L. monocytogenes isolates were obtained from the Medicinal and Aromatic Plants Department, Natural Products Unit, Desert Research Center, Mathaf El-Matarya St., El-Matareya, Cairo 11,753, Egypt, by Dr. Gamal Osman Hassan.

Lactic acid and probiotic bacterial strains

Five lactic acid bacteria (LAB) and probiotic strains were sourced from international culture libraries. Table 1 lists the strains’ suppliers, along with the precise temperature and aeration level required for each strain. These strains were selected for their frequent use in the food industry or for information on their impact on food mutagens. Agar plates made with deMan, Rogosa, and Sharpe (MRS) media (Difco Laboratories) were used to cultivate the bacteria. Bacteria were grown on MRS agar plates (Difco Laboratories, deMan, Rogosa, and Sharpe). Anaerobic strains were kept in Oxoid and Anaerogen jars. Five LAB and probiotic strains were sourced from international culture libraries.

Table 1.

Sources of bacterial strains.

Strain	Source	Oxygen requirement
B. lactis Bb12	1	Anaerobic
L. acidophilus ATCC 20,552	2	aerobic
L. rhamnosus TISTR 541	3	Aerobic
B. angulatum DSMZ 20,098	4	Anaerobic
L. plantrium	2	Aerobic

(1) Chr. Hansen-Denmark.

(2) Egyptian Microbial Culture Collection (EMCC) at Cairo Microbiological Resources Center (Cairo, MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt.

(3) Thailand Institute of Scientific and Technological Research, Bangkok, Thailand.

(4) German Center for Culture Collection (DSMZ-Germany).

Until they were needed, fully developed colonies were kept on plates at 4 °C and subjected to monthly subculturing; using 90% glycerol as a cryoprotectant, cell suspensions or spores were cryopreserved for long-term storage at -80 °C in cryoflasks. Lactobacilli and, with the addition of cysteine, bifidobacteria were cultivated in MRS broth. The cultures were then incubated at the optimal growth temperature for a full day.

Juice extracting and composition

Biswas et al.⁴ reported the steps involved in producing goldenberry juice. Only fruits with comparable maturity stages were chosen for processing. The whole berries were pasteurized at 80 °C for 10 min to deactivate the endogenous enzymes after being processed for 5 min in a Waring blender (Moulinex Ovatio 3, France). The pulp mixtures were allowed to cool to room temperature following heat treatment. The Pectinex 3XL enzyme (Novozyme Corp., Bagsvaerd, Denmark) was added at a utilization level of 0.0827 mL/kg. Following an hour at room temperature, the must was compressed in a 70-L Enrossi bladder press (Enoagricol Rossi, Calzolaro, Italy) using screens and press cloths. A Flowjet Model T2913 with 9–10 mm filter pads (Buon Vino Inc., Cambridge, Ontario, Canada) was utilized to filter the juice. Once the juice was sealed in sterile glass jars, it was stored at 4 °C.

Following the methods reported by Ramadan and Moersel³⁴, chemical composition (e.g., moisture, sugars, protein), physicochemical traits (pH, acidity, total soluble solids), and bioactive compounds were analyzed in the juice using standard analytical methods (AOAC, spectrophotometry, chromatography). All measurements were conducted in triplicate to ensure reliability.

Assay of pathogenic microorganisms

Agar plates were used to cultivate bacterial strains overnight at 37 °C under aerobic conditions. After that, the bacteria were collected in a biosafety cabinet using a sterile 10-milliliter loop and put in 10 milliliters of phosphate-buffered saline (PBS). The optical density (OD) of the bacterial culture was adjusted to an absorbance value of 0.10 at 600 nm using a T80 = UV/VIS spectrophotometer (PG Instruments LTD). An aliquot of 100 mL of bacterial suspension was added to 900 mL of broth (LB broth for E. coli and S. sureus, and BHI broth for L. monocytogenes), skim milk, or whole milk in a sterile 24-well cell culture plate. Both skim and whole milk were purchased from a local store in Sadat City and subsequently UV-sterilized in a biosafety cabinet. The wells were incubated at 37 °C for 0, 24, 48, and 72 h after each well was filled with either 100 mL of 10% goldenberry juice or the same volume of sterile deionized water (control)⁴. After incubation, serial dilutions were performed in PBS. Bacterial colony-forming units (CFU) were measured by plating serial dilutions of E. coli, S. aureus, and L. monocytogenes on LB, BHI, and MacConkey agar, respectively. For each dilution in each study, triplicate plates were used. Three trials were carried out for each microbe.

Lactic acid and probiotic strains growth and survival in goldenberry juice

B. angulatum DSMZ 20,098 and B. lactis Bb12 were grown on MRS agar plates overnight (EMD, Rockland, MA) at 37 °C in anaerobic conditions. Cells were then collected in 10 mL of phosphate buffer solution PBS using a sterile 10 mL loop under a biosafety cabinet. L. acidophilus ATCC20552, L. plantarum, and L. rhamnosus TISTR 541 were grown on MRS agar plates overnight in aerobic conditions. A T80 = UV/VIS spectrophotometer (PG Instruments LTD) was used to calibrate the bacterial suspension’s optical density (OD) to an absorbance of approximately 0.10 at 600 nm. An aliquot of 100 mL of bacterial suspension was added to each well of a sterile 24-well cell culture plate containing 900 mL of either blackberry juice or MRS broth. After that, the plate was incubated for several periods of time at 37 °C (0, 24, 48, and 72 h)⁴. Following incubation, successive dilutions were made in PBS and plated on MRS agar to count the number of bacterial colony-forming units (CFU). For each dilution in each study, triplicate plates were used. Three experiments were conducted for each microbe.

Statistical analysis

Bacterial growth and survival were evaluated by comparing viable cell counts at 0, 24, 48, and 72 h between treatment and control groups. Control samples consisted of milk or broth media without goldenberry juice supplementation. All experiments were performed in replicates, and results were expressed as mean ± standard deviation. Statistical analyses were conducted to evaluate the effects of treatment, storage time, medium type, bacterial strain, and their interactions on bacterial growth and survival responses. Data were analyzed using analysis of variance (ANOVA), followed by Tukey’s multiple comparison test to determine significant differences among treatments and sampling times at P < 0.05. Pearson’s correlation analysis was additionally performed using SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA) to assess relationships among bacterial growth and survival parameters.

Results and discussion

Juice composition and properties

According to our results, goldenberry juice had a high moisture content (93 g/100 g), relatively low protein (0.46 g/100 g) and lipid (0.29 g/100 g) content, and moderate carbohydrate (5.70 g/100 g) content, with total sugars around 5.1 g/100 g. The juice was naturally acidic, with a pH of 3.84 and titratable acidity of 0.93% (as citric acid), while total soluble solids measured 10.5 °Brix. It also contained moderate pulp content (3.78 g/100 g). The juice is rich in vitamin C (47 mg/100 g) and contains measurable phenolics (6.5 mg/100 g). These constituents contributed to strong antioxidant activity.

Previous reports³⁴ on Physalis peruviana highlighted that the juice is characterized by high soluble solids (~ 15%), total sugars (~ 4.9 g/100 g), organic acids (0.9-1.0%), and low pH (3.79–3.86), contributing to its stability and sensory quality^34,35. Physalis peruviana juice is a rich source of vitamin C (~ 46 mg/100 g), carotenoids (notably β-carotene), and phenolic compounds, with quercetin, kaempferol, and myricetin identified as major flavonoids responsible for strong antioxidant capacity^35,42. The juice contained approximately 0.2% oil, composed mainly of linoleic, oleic, palmitic, and γ-linolenic acids, as well as phytosterols (Δ5-avenasterol and campesterol) and tocopherols (γ- and α-forms), thereby enhancing its nutritional and functional value^35,40. Withanolides and other bioactive phytochemicals further support its anti-inflammatory and health-promoting properties³⁵.

As shown in Fig. 1 (A, B, C, and D), the addition of goldenberry juice to the MRS broth markedly reduced the bacterial counts of all tested pathogenic strains throughout the incubation period. At the beginning of incubation (0 h), all treatments showed similar initial counts, but differences emerged after 24 h. For E. coli, the viable count in the control MRS broth reached 9.6 log CFU/mL, while the medium containing goldenberry juice recorded only 6.5 log CFU/mL, indicating a reduction of about 3.1 log units. This inhibitory effect became more pronounced over time, as the counts at 72 h declined to 6.1 in the control versus 4.1 in the supplemented medium, indicating approximately a 2-log reduction in the presence of the extract.

Fig. 1.

Inhibition of the growth of pathogenic microorganisms using of broth and golden berry juice, in selective broth and selective broth with 10% golden berry juice at 0, 24, 48, and 72 h. (A) Escherichia coli, (B) Pseudomonas aeruginosa, (C) Staphylococcus aureus, (D) Listeria monocytogenes. Different letters within the same column indicate significant differences among storage periods according to Tukey’s test (P < 0.05).

A similar pattern was observed for P. aeruginosa, with counts at 72 h decreasing from 6.4 log CFU/mL in the control to 4.3 log CFU/mL in the presence of goldenberry juice. S. aureus and L. monocytogenes showed comparable reductions, with final counts at 72 h declining from 7.4 to 6.4 in the control to 4.9 and 4.1 log CFU/mL, respectively. These consistent reductions across all strains confirm that the goldenberry extract exerted a broad-spectrum antimicrobial effect, reducing bacterial growth by roughly 2–3 log units during the 72-h incubation period. The quantitative results demonstrate that bacterial populations continued to decline with prolonged incubation, suggesting that the extract’s antimicrobial activity is both stable and time-dependent. This sustained inhibition may be attributed to the cumulative action of bioactive compounds such as phenolic acids, flavonoids, and withanolides, which can damage bacterial membranes, alter intracellular enzyme systems, and interfere with nutrient uptake. Among the tested microorganisms, the inhibitory effect was relatively more pronounced against E. coli and L. monocytogenes, which might be related to differences in cell wall structure, sensitivity to oxidative stress, or metabolic adaptability. Gram-negative bacteria such as E. coli and P. aeruginosa possess outer membranes that typically reduce susceptibility to natural antimicrobials; however, the results suggest that the bioactive compounds in goldenberry juice remained effective in suppressing their growth, possibly through oxidative or enzymatic mechanisms.

Overall, the data clearly indicate that incorporating goldenberry juice into the growth medium significantly reduced bacterial proliferation compared to the control, confirming the potent antimicrobial potential of P. peruviana fruit and supporting its potential use as a natural antimicrobial agent in food and pharmaceutical applications.

Figure 2 (A, B, C, and D) presents a comparative evaluation of the growth and survival patterns of four pathogenic bacteria (E. coli, P. aeruginosa, S. aureus, and L. monocytogenes) in skim milk and whole milk, both with and without 10% goldenberry juice, at incubation periods of 0, 24, 48, and 72 h. The data provide clear evidence that the addition of goldenberry juice consistently reduced bacterial growth rates across all species and incubation times. For E. coli, the control samples (skim and whole milk without juice) showed an increase in bacterial counts from 6.7 to 6.4 log CFU/mL at 0 h to 9.6 and 8.6 log CFU/mL after 24 h, respectively. These values remained high up to 48 h (9.3 and 9.7 log CFU/mL), indicating active growth. However, when 10% goldenberry juice was incorporated, the bacterial load was notably lower: 8.1 log CFU/mL at 24 h and decreased further to 6.6 and 6.1 log CFU/mL at 48 and 72 h, respectively. This represents an approximate 2.5-3.0 log reduction compared with the control, demonstrating a pronounced inhibitory effect on E. coli proliferation.

Fig. 2.

Comparison of the growth and survival of pathogenic microorganisms in skim milk, whole milk with and without 10% golden berry juice at incubation periods of 0, 24, 48, and 72 h. (A) Escherichia coli, (B) Pseudomonas aeruginosa, (C) Listeria monocytogenes, (D) Staphylococcus aureus. Different letters within the same column indicate significant differences among storage periods according to Tukey’s test (P < 0.05).

A similar inhibitory trend is evident for P. aeruginosa, which grew from 7.6 to 9.7 log CFU/mL in skim milk within 24 h, whereas the juice-supplemented samples reached only 7.3–7.4 log CFU/mL at the same time point. After 72 h, bacterial counts in the control remained high (around 8.3–8.5 log CFU/mL), whereas those in goldenberry-treated milk declined to 5.8-6.0 log CFU/mL. This roughly 2.5 log difference indicates that goldenberry juice effectively suppressed this resilient Gram-negative bacterium, possibly by disrupting its outer membrane through the organic acids and polyphenols present in the extract. S. aureus also displayed marked sensitivity to goldenberry juice. The bacterial count in skim milk alone increased from 9.4 to 9.2 log CFU/mL after 24 h, while the juice-containing samples showed significantly lower counts (6.0-6.1 log CFU/mL). After 72 h, the difference became even more pronounced, with the treated samples recording only 5.1 log CFU/mL compared to 8.3 in the untreated milk. This nearly 3-log reduction strongly supports the juice’s potent inhibitory activity against Gram-positive bacteria, likely due to the combined effects of low pH, antioxidant compounds, and potential interference with cell wall synthesis. Regarding L. monocytogenes, a similar behavior was recorded. In the absence of goldenberry juice, counts reached 9.3 log CFU/mL after 24 h and remained around 8.8–8.9 by 72 h. Conversely, the addition of the juice resulted in reduced counts of 8.1 log CFU/mL at 24 h and a consistent decline to 6.2 and 5.3 log CFU/mL by 72 h. This reduction of nearly 3.5 logs demonstrates that the antimicrobial effect of goldenberry is not only maintained but possibly enhanced during prolonged incubation, suggesting that its active components remain stable over time.

Collectively, the results indicate that goldenberry juice significantly inhibits bacterial growth in both skim and whole milk, with greater effectiveness at longer incubation times. The inhibitory action was more pronounced against S. aureus and L. monocytogenes compared with the Gram-negative species. These results confirm that the juice’s natural phytochemicals and acidity create unfavorable conditions for bacterial survival, potentially extending the microbial stability and safety of dairy products.

The overall findings indicate that goldenberry extract possesses broad-spectrum antimicrobial activity that remains effective in both direct culture conditions and in food matrices, such as milk. The initial results from the direct exposure assays confirmed that the extract exhibited measurable inhibitory zones against all tested pathogenic strains, including E. coli, S. aureus, P. aeruginosa, and L. monocytogenes. The strength of inhibition varied by bacterial type, with S. aureus and L. monocytogenes showing greater sensitivity than the Gram-negative bacteria. These differences suggest that the phenolic and flavonoid constituents of goldenberry can interfere more efficiently with the cell wall structure and metabolic pathways of Gram-positive organisms.

When the extract was incorporated into milk systems, a consistent inhibitory pattern was observed, reinforcing the antimicrobial capability of goldenberry under realistic food conditions. Although the milk environment provides nutrients that typically support microbial growth, the presence of goldenberry juice markedly reduced bacterial counts throughout the incubation periods. This demonstrates that the active compounds remain stable and functional in complex matrices and that their activity is not limited to in vitro assays. The gradual decrease in viable counts over time, particularly evident at 48 and 72 h, reflects a time-dependent inhibitory mechanism that may involve cumulative damage to bacterial cells or interference with their replication. The data highlight two important conclusions: first, goldenberry juice shows antimicrobial and growth-inhibitory effects; and second, these effects are retained and even enhanced when applied within dairy systems, making it a promising natural preservative for improving the microbial safety of milk-based products. Overall, the study supports the potential use of goldenberry as a functional bio-ingredient for food preservation and pathogen control.

Impact of goldenberry juice on the growth of lactic acid and probiotic bacteria

The effects of goldenberry juice on the growth of lactic acid bacteria and probiotic strains (L. acidophilus ATCC 20552, L. plantarum, L. rhamnosus TISTR 541, B. lactis Bb12 and B. angulatum DSMZ 20098) are shown in Fig. 3 (A, B, C, D, and E). We observed that the effect of goldenberry juice on Lactobacillus is species-specific and probiotic bacteria. All five lactic acid bacteria probiotic strains exhibited distinct growth patterns in MRS broth supplemented with goldenberry juice.

Fig. 3.

Effect of golden berry juice on the growth of Lactobacillus and Bifdobacterium strains in MRS broth and MRS broth with 10% blackberry juice at incubation time of 0, 24, 48, and 72 h. (A) Lactobacillus acidophilus ATCC 20,552, (B) Lactobacillus rhamnosus TISTR 541, (C) Lactobacillus plantarum, (D) Bifdobacterium lactis Bb12, (E) Bifdobacterium angulatum DSMZ 20,098. Different letters within the same column indicate significant differences among storage periods according to Tukey’s test (P < 0.05).

The growth of L. rhamnosus TISTR 541 was significantly (P < 0.05) stimulated (> 2 logs CFU/mL) by goldenberry juice at all three time points (24, 48, and 72 h) as compared with the growth in MRS broth alone. For L. plantarum, L. acidophilus, B. lactis Bb12, and B. angulatum DSMZ 20,098, 10% supplemented goldenberry juice in MRS broth significantly promoted growth only at the 48-h time point. These results revealed that goldenberry juice significantly stimulated (P < 0.05) the growth of lactic acid bacteria in a time-dependent manner for some species.

Figure 4 (A, B, C, D, and E) shows the growth of L. acidophilus ATCC 20,552, L. plantarum, and L. rhamnosus TISTR 541 in both skim and whole milk with or without 10% goldenberry juice. We found that the growth of L. plantarum was significantly stimulated (2.74–4.20 logs CFU/mL) by goldenberry juice in both skim and whole milk at all time points (24, 48, and 72 h). Compared with skim milk, whole milk supplemented with 10% goldenberry juice had a stronger stimulatory effect on the growth of L. plantarum. Both skim and whole milk supplemented with goldenberry juice stimulated the growth of L. acidophilus ATCC 20,552, L. rhamnosus TISTR 541, and L. plantarum less efficiently (2.5-3.0, 2.41–3.3 and 0.77–2.24 logs CFU/mL for L. acidophilus ATCC 20552, L. rhamnosus TISTR 541, and L. plantarum, respectively) compared to B. lactis Bb12 and B. angulatum DSMZ 20,098.

Fig. 4.

Comparison of growth and survival of Lactobacillus and Bifdobacterium strains in skim milk, whole milk with and without 10% golden berry juice at incubation periods of 0, 24, 48, and 72 h. (A) Lactobacillus acidophilus ATCC 20,552, (B) Lactobacillus rhamnosus TISTR 541, (C) Lactobacillus plantarum, (D) Bifdobacterium lactis Bb12, (E) Bifdobacterium angulatum DSMZ 20,098. Different letters within the same column indicate significant differences among storage periods according to Tukey’s test (P < 0.05).

Effect of goldenberry juice on the correlations of growth of pathogenic lactic acid and probiotic bacteria

A Pearson correlation analysis was performed among these bacteria, including pathogens, lactic acid bacteria, and probiotic bacteria, to understand their relationships better. Tables S2 and S3 show the correlation matrices of survival and growth of bacteria (in broth and milk) without and with 10% goldenberry juice, respectively. After the addition of goldenberry juice, the correlation coefficients were significant among the four pathogens and the five lactic acid bacteria and probiotic bacteria, but there was no statistically significant correlation between pathogen species and Lactobacillus species (P > 0.05). After the addition of goldenberry juice, the correlation coefficients were significant among the four pathogens and the five Lactobacillus and probiotic bacteria, but there was no statistically significant correlation between pathogen species and Lactobacillus species (P > 0.05).

The statistically significant differences observed among treatments and storage periods suggest selective responses of pathogenic and probiotic bacteria to goldenberry juice supplementation, indicating potential biological relevance beyond simple growth variation.

The correlation analysis presented in this study should be interpreted cautiously, as the observed relationships may partly reflect common time-dependent trends rather than direct biological interactions among microbial species. Therefore, more advanced multivariate analyses may provide deeper insight into microbial response patterns in future studies.

The clustered heatmap analysis revealed overall similarity in the responses of pathogenic and probiotic bacteria under the tested conditions. Strong positive correlations were observed among several bacterial groups, particularly within probiotic strains and among certain pathogenic bacteria. However, these associations should be interpreted cautiously, as they may partly reflect common time-dependent response patterns rather than direct biological interactions. Therefore, the heatmap was primarily used as an exploratory visualization tool to summarize general trends in microbial responses (Fig. 5). The clustered heatmap analysis revealed distinct response patterns among pathogenic and probiotic bacteria in the presence of 10% goldenberry juice. Strong positive correlations were observed among several probiotic strains, particularly L. acidophilus, L. plantarum, L. rhamnosus, and B. lactis, suggesting relatively similar growth responses under the tested conditions.

Fig. 5.

Clustered heatmap visualization of Pearson correlation coefficients among pathogenic and probiotic bacteria under experimental conditions without goldenberry juice supplementation. Color intensity reflects the strength of the correlation coefficient (r), while hierarchical clustering illustrates patterns of similarity among bacterial responses. *, **, and *** indicate statistical significance at P < 0.05, P < 0.01, and P < 0.001, respectively.

In contrast, weak or negative correlations were observed between some pathogenic and probiotic bacteria, indicating differential microbial responses following goldenberry juice supplementation. However, these associations should be interpreted cautiously, as they may partly reflect common time-dependent response trends rather than direct biological interactions. The heatmap, therefore, served primarily as an exploratory visualization tool to summarize overall microbial response patterns (Fig. 6).

Fig. 6.

Clustered heatmap visualization of Pearson correlation coefficients among pathogenic and probiotic bacteria in the presence of 10% goldenberry juice. Color intensity represents the strength and direction of Pearson correlation coefficients (r) among bacterial responses under experimental conditions supplemented with 10% goldenberry juice. Hierarchical clustering was used to illustrate similarity patterns among pathogenic and probiotic bacteria. Warm colors represent positive correlations, whereas cool colors represent negative correlations. *, **, and *** indicate statistical significance at P < 0.05, P < 0.01, and P < 0.001, respectively.

Due to changes in human populations’ eating patterns and means of subsistence, the incidence of diseases acquired from contaminated food has substantially increased in recent years^43–45. More than half of foodborne infections are caused by the principal causative agents Salmonella, L. monocytogenes, and E. coli O157:H7. For the food sector and public health organizations, managing these foodborne enteric bacteria is a serious challenge. Furthermore, it is now harder to guarantee the safety of the food supply chain due to the comeback of foodborne bacteria that are resistant to drugs^1,46–54.

A viable alternative to managing foodborne bacterial infections is the use of natural antimicrobials. Research on the potential antimicrobial role of natural products, such as berries and other fruits and vegetables, is still in its infancy. One area of interest is the possible role of antimicrobial plant products in inhibiting the growth and preventing cross-contamination of foodborne bacterial pathogens. The effects of blackberry juice are in contrast to blueberry juice, which displayed no stimulating effects on the growth of probiotic bacteria^4,55.

The present study demonstrated with strong evidence that a small amount of goldenberry juice (10%) has strong inhibitory effects against several foodborne pathogens and stimulatory effects on the growth of beneficial bacterial species. We have demonstrated in this study that goldenberry juice significantly inhibited the growth of pathogenic bacteria, including E. coli, S. aureus, L. monocytogenes, and P. aeruginosa (2–4 logs CFU/mL in broth and 1–2 logs CFU/mL in milk). Phenolic components may be responsible for this inhibitory effect. This is confirmed by recent research showing that phenolic compounds, notably the ellagitannins in berries, are powerful inhibitors of S. enterica and L. monocytogenes^18,56. In their reports, they also found that phenolic compounds, especially ellagitannins in berries, were strong inhibitors of S. enterica and L. monocytogenes. Previous research has also shown that blueberry juice inhibits the growth of E. coli O157:H7, S. typhimurium, L. monocytogenes, and Campylobacter jejuni⁴.

The partial hydrophobicity of proanthocyanidins, anthocyanins, and phenolics may be responsible for this suppression, as it enables them to adhere to the bacteria’s outer membrane and alter its fluidity⁵⁷. The mechanism of inhibition may involve this interaction at the membrane contact site^51,52,58. Numerous studies have demonstrated that the presence of phenolic compounds in berries does not limit the growth of probiotic bacteria^{4,17,18,51,52,54}.

Numerous studies^4,18,51,52 have demonstrated that the presence of phenolic chemicals found in berries does not limit the growth of probiotic bacteria. 10% goldenberry juice greatly accelerated the growth of the probiotic and lactic acid species utilized in this investigation (about 1–4 logs CFU/mL in milk and 1.0-2.5 logs CFU/mL in MRS).

The stimulatory effect of goldenberry juice on probiotic bacteria may be related to the availability of fermentable sugars and other bioactive constituents that could support bacterial growth and metabolism. The wide range of dietary substrates utilized by probiotic bacteria may contribute to this stimulation. In particular, the rhamnosidase-producing activity reported for Lactobacillus strains may facilitate the utilization of phytochemical compounds present in goldenberry juice^{18,33,56,59,60}. In addition, the slightly acidic conditions and sugar content of goldenberry juice may favor the growth of lactobacilli by serving as accessible carbon sources^61,62. However, these proposed mechanisms were not directly investigated in the present study, and further metabolic, enzymatic, and molecular analyses are required to clarify the pathways responsible for the selective enhancement of probiotic microorganisms.

The fact that milk and broth have diverse media compositions may contribute to their disparate impacts. Many berries, including chokeberries (A. melanocarpa), blueberries, blackberries, raspberries, bilberries, and black currants, had high total antioxidant content⁶³. Blueberries demonstrated the highest overall antioxidant capacity when measured by DPPH· and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), with a TEAC value of 15 mM Trolox/100 g dry weight (DW). According to Huang et al.⁶⁴, blueberries also exhibited the greatest total anthocyanidin concentration (24 mg catechin/g DW), total flavonoid content (36 mg rutin/g DW), and total phenolic content (9 mg gallic acid/g DW).

The aforementioned study reports that the immature wild blueberry (Vaccinium stenophyllum Steud.) fruit extract exhibited the highest content of total phenolic compound (19.153 mg gallic acid equivalent /g DW) and the highest antioxidant activity by both ABTS (196.761 M Trolox equivalent/g DW) and DPPH assays (146.580–6.466 M Trolox equivalent/g DW). On the other hand, the mature blueberry fruit extract displayed the highest content of anthocyanins (0.141 − 0.004 mg cyanidin-3-glucoside equivalent/g DW) and cyanidin-3-glucoside (19.230–0.309 mg cyanidin-3-glucoside equivalents/g DW)⁶⁵.

Correlation coefficients from the Pearson analysis indicated that the addition of 10% goldenberry juice altered the growth and survival patterns of lactobacilli and pathogens. In the absence of goldenberry juice, every bacterial strain expanded without any hindrance. As a result, Table S2 shows substantial correlation coefficients for most microorganisms. The correlation coefficients among the groups supplemented with goldenberry juice were statistically significant (P < 0.05) due to the similarity in the trends of inhibition or stimulation across four pathogens, five lactobacillus species, and probiotics (Table S3).

However, the relationship between the pathogens and the lactic acid- and probiotic bacteria was not statistically significant because of the latter’s both encouraging and inhibitory effects on the pathogens. According to a prior study^4,54,66,67, adding blueberry juice to skim milk reduced the growth of foodborne bacterial pathogens but did not affect L. bulggaricus. In this work, we demonstrated that the addition of 10% goldenberry juice significantly suppressed the growth of all pathogenic bacterial strains tested, in both skim milk and whole milk. Interestingly, we also observed that when goldenberry juice was added to milk and broth media, all strains of Lactobacillus and probiotics grew noticeably faster. Therefore, goldenberry juice has the potential to serve as both a growth promoter for probiotics and Lactobacillus, two beneficial bacteria, and a promising natural antibiotic against foodborne bacterial infections. Since many chemical antibacterial agents are prohibited for use in organic foods due to stringent regulations, this information is particularly essential for organic foods. It’s crucial to consider potential effects on beneficial microbes when using antibiotics to treat illnesses. Antibiotic treatments typically reduce the normal gut flora, promoting the growth of highly pathogenic bacteria⁵².

Goldenberry juice can greatly stimulate the proliferation of probiotic species, as our study has shown. This conclusion has been clarified by earlier studies by Pereira et al.,⁶², Demir et al.⁶¹, and Shan et al.⁶⁸. Pereira et al.⁶² reported that L. casei growth increased by 0.52 log CFU/mL following 16 h of incubation at 30 °C with cashew apple juice. After that, it rose by 0.3 log CFU/mL for a total of 28 days at 4 °C. Demir et al.⁶¹ found that carrot juice had a similar effect on the development of L. plantarum. The mechanisms by which goldenberry juice promotes the growth of probiotic lactic acid bacteria and prevents specific foodborne pathogens remain unclear and require further research.

The selective stimulation of probiotic bacteria observed in the present study may be associated with the bioactive composition of goldenberry juice, particularly its phenolic compounds and fermentable constituents. Recent studies have demonstrated that plant-derived phenolics, polysaccharides, and oligosaccharides can modulate microbial growth, fermentation behavior, and metabolic activity during food fermentation and gut microbiota interactions^69–71. Similar observations have been reported for berry-derived bioactive compounds, which enhance the growth and metabolic adaptation of beneficial microorganisms by improving substrate utilization and microbial responses. The fermentation of blackberry polysaccharides promoted the increase of Bacteroidetes and the decrease of Firmicutes. These findings may partially explain the enhanced growth responses of probiotic strains observed following goldenberry juice supplementation in the present study.

The antimicrobial activity observed in the present study may be associated with the combined effects of phenolic and flavonoid compounds, acidity, organic acids, sugars, and processing-related changes rather than a single bioactive component. Previous studies have suggested that phenolic constituents of goldenberry may contribute to membrane disruption and oxidative stress in bacterial cells. However, the exact mechanisms underlying the selective inhibition of pathogenic bacteria and stimulation of probiotic microorganisms remain unclear. Therefore, further investigations using pH-adjusted, neutralized, fractionated, and phenolic-depleted juice systems, as well as mechanistic and comparative analyses with conventional antibiotics and food preservatives, are recommended to elucidate the relative contributions of each factor better and to evaluate the practical potential of goldenberry juice as a natural antimicrobial ingredient in food applications.

Although goldenberry juice demonstrated significant antimicrobial effects against pathogenic bacteria in inoculated milk systems under controlled experimental conditions, the present study is insufficient to definitively distinguish between bacteriostatic and bactericidal modes of action or to fully confirm its potential to extend shelf life in dairy applications. Therefore, further investigations involving MIC and MBC determination, time-kill kinetics, membrane integrity and re-growth analyses, as well as refrigerated storage studies including natural microbiota, total viable count, yeast and mold growth, pH and acidity changes, sensory evaluation, color and flavor acceptability, and comparisons with conventional dairy preservatives are necessary to characterize better the antimicrobial properties and practical applicability of goldenberry juice in food systems.

Conclusions

Antibiotics have been widely used to reduce pathogen infections for over 90 years. Controlling and treating various infectious diseases in humans has become increasingly challenging in recent decades due to the rise in antibiotic-resistant bacteria. Plant phytochemicals are becoming increasingly popular as alternative treatments for bacterial infections. Fruit extracts contain a variety of useful ingredients, including bioactive antioxidant compounds. We now know more about how these chemicals’ bioactivity can be employed to treat and prevent infections in humans, and they have several recognized antibacterial mechanisms. Acting on a single ingredient, as is typically utilized in antibiotic therapy, may delay the development of bacterial resistance, making fruit extracts an attractive alternative to antibiotics.

Suggesting that fruit extracts may also preserve probiotic organisms in the microbiome. To clarify the mechanisms of action of the phytochemicals in these combinations, further investigation is still required. Furthermore, the majority of research assessing phytochemical efficacy relies on simple in vitro models, and results can differ significantly from those observed in the complex conditions of the gastrointestinal tract. In addition to being studied in animal models, artificial digestive models should be evaluated and shown to be beneficial for human health.

Supplementary Information

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Author contributions

Mohammed Aladhadh: Methodology, Investigation, Applications, Writing-review & editing. Rafaat M. Elsanhoty: Formal analysis, writing review, and editing. Mahmoud A. Al-Saman: Data curation, Methodology, Formal analysis, Investigation. Mohamed Fawzy Ramadan: Conceptualization, Supervision, Writing-review & editing. Hafsa Nour El-Din Abd El-Kader Ebrahim: Formal analysis, Conceptualization, Supervision, Writing-review & editing Rushdy M. A.: Formal analysis, Conceptualization, Supervision, Writing-review & editing Fatma Ibrahim Abou-Elazm: Conceptualization, Supervision, Writing-review & editing. All authors read and approved the final manuscript.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Competing interests

The authors declare no competing interests.

Consent for publication

All authors consent for the manuscript to be published.