3.1 In vitro studies on the anti-bacterial effects of Aronia melanocarpa

In their study, Nowak et al. [12] evaluated the anti-microbial activities of distinct fruit juices including black chokeberry juice. Following neutralization, the analysis of the black chokeberry juice by the diffusion assay revealed a moderate anti-microbial activity against distinct strains of Gram-positive bacterial species, such as Staphylococcus aureus, Listeria monocytogenes, Listeria innocua, and Bacillus cereus, as opposed to Enterococcus faecalis and Clostridium perfringens. In contrast, no anti-microbial activity could be assessed against the tested strains of Gram-negative bacterial species, namely Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Salmonella Enteritidis, Pseudomonas aeruginosa, and Pseudomonas fluorescens. The authors suggested that black chokeberry juice among other fruit juices might be considered as promising natural food preservative given its anti-microbial effects directed against Gram-positive food-poisoning bacterial species such as Listeria or B. cereus [12].

Raudsepp et al. [13] tested the anti-bacterial effects of black chokeberries against food-spoiling bacteria. The analyses of 20% and 96% ethanolic plant infusions directed against Gram-positive species applying the agar diffusion assay revealed anti-microbial effects against the B. cereus reference strain ATCC 11778 even at 1:20 and 1:40 dilutions. However, only 96% ethanolic infusions were effective against Bacillus pumilus CV 607, Kocuria rhizophila, and L. monocytogenes ATCC 13929. Interestingly, anti-bacterial activities could also be detected against distinct Gram-negative isolates of S. Enteritidis ATCC 13076, and E. coli NCCB 100282 (for 96% ethanolic infusions), whereas the black chokeberry extract at both ethanolic concentrations resulted in growth inhibition of Campylobacter jejuni ATCC 33291. Hence, ethanolic black chokeberry infusion did not only exert anti-microbial activities against distinct Gram-positive, but also Gram-negative food-spoiling bacterial species [13].

The anti-bacterial effects of Aronia melanocarpa anthocyanins (AMA) against E. coli 0157:H7 were studied by Deng and colleagues [14]. To elucidate the mechanism underlying potential anti-bacterial effects of AMAs directed against E. coli 0157:H7 at the protein level, the authors applied the tandem mass tag quantitative proteomics approach in combinations with multiple reaction monitoring. The findings demonstrated that 628 of the 1739 proteins (262 of which were down-regulated and 366 of which up-regulated) were changed in E. coli upon AMA treatment. In depth bioinformatics analysis further revealed that these differentially expressed proteins interacted in distinct biochemical pathways involved in oxidative stress response, peptidoglycan production, DNA replication and repair, as well as E. coli protein biosynthesis and homeostasis. In consequence, AMA challenge exerts bactericidal effects leading to distinct morphological alterations of the bacterial cells ultimately resulting in cell death as evidenced by electron microscopy [14].

In another study, the same research group [15] discovered compelling reactions of bacterial mechanosensitive channels when exposed to AMA. The authors observed that the activation of small-conductivity mechanosensitive channels (MscL) and of low conduct mechanosensitive channels (YnaI) in E. coli 0157:H7 cells decreased upon AMA challenge, suggesting a protective reaction of the bacterial cells that facilitated cell entry of AMA resulting in elevated intracellular reactive oxygen species (ROS) levels. Furthermore, AMA exposure down-regulated the expression of heat shock proteins and chaperons like Hs1R, Hs1sO, HtpX, HtpG, which could lead to a compromized stress response of the bacterial cells mounting in cell death [15].

In their study, Bräunlich et al. [16] tested the anti-biofilm-forming activity of defined Aronia melanocarpa constituents against both, Gram-positive and Gram-negative bacteria. Therefore, B. cereus 407 and E. coli K12 JM109 as well as the uropathogenic E. coli ATCC 700928 strain, respectively, were incubated with distinct crude Aronia extracts, sub-fractions, and compounds at concentrations of 1 mg mL⁻¹ each in a microtiter plate assay. The incubation time for B. cereus and E. coli were 48 h and 24 h, respectively, to assure maximum biofilm production. Overall, most of the Aronia samples exhibited biofilm inhibition against B. cereus of differing activities, but to a much lesser extent against the E. coli strains. When defined Aronia melanocarpa constituents were studied for their inhibitory activity on biofilm formation, it was observed that epicatechin and cyanidin 3-xyloside showed the highest activity against biofilm formation by E. coli K12 JM109, whereas epicatechin displayed no such inhibitory effect at all in case of the tested B. cereus 407 strain. When 1.0 mg of 50% ethanolic Aronia berry extract was tested for its anti-bacterial activity against B. cereus 407, however, the growth of the Gram-positive strain could be inhibited (with a median inhibition zone diameter of 10.2 ± 0.8 mm), which was, however, still less effective if compared to a control culture exposed to 10 µg gentamicin resulting in an inhibition zone diameter of 20.5 ± 0.7 mm. Interestingly, the anthocyanin cyanidin 3-xyloside resulted in a 50% decrease of B. cereus 407 biofilm formation, whereas the other anthocyanins, such as cyanidin 3-galactoside, cyanidin 3-glucoside, and cyanidin 3-arabinioside even enhanced the biofilm production by more than 350%. Nevertheless, the growth of the E. coli K12 JM109 and the uropathogenic E. coli ATCC 700928 strains was not compromised upon challenge with respective Aronia compounds [16].

Another study [17] tested extracts of Aronia leaves (in 80% ethanolic infusion) against Gram-positive bacteria such as B. cereus ATCC 14579 and L. innocua KTCC 506 and Gram-negative bacteria including E. coli 0157:H7 43895 and S. Typhimurium DT 104. The Aronia leaves used for the extraction of cultivars were gathered at three different growth states (namely, young, harvest, and old) and were examined by the disk diffusion test and spot-on-the-lawn method. The results showed that only cultivars from two of the old Aronia leaf extracts displayed anti-bacterial activities exclusively against B. cereus. When assessing the growth curves in liquid media, B. cereus showed a lag time of approximately 2 h and grew up to 5.74 log₁₀ colony-forming units (CFU) per mL after 6 h in the control culture without extract. Upon co-incubation with the two extract cultivars in the liquid media, less bacterial growth of the Gram-positive bacteria could be assessed in both cases, whereas one cultivar even did not show any growth after 5 h of co-incubation. Notably, none of the cultivars of all different harvest states exerted anti-bacterial activity against L. innocua and the Gram-negative strains E. coli O157:H7 and S. Typhimurium [17].

Efenberger-Szmechtyk et al. [18] examined the anti-bacterial effects of Aronia melanocarpa leaf extracts directed against typical meat spoiling bacteria. The analyses revealed that the leaf extracts were able to both, decrease the growth rate and lengthen the lag phase of the tested Gram-positive bacteria such as S. aureus and Brochotrix thermospacta, whereas the tested L. monocytogenes ATCC 13992 bacteria were resistant. The Aronia melanocarpa leaf extract was also effective against Gram-negative bacteria as shown for a Salmonella enterica isolate from chicken meat [18].

In a follow-up study by the same research group [19], the authors explored the mechanisms underlying the anti-bacterial effects of Aronia melanocarpa leaf extracts against food spoilage bacteria. Overall, the bacterial viability compromising effects of the leaf extracts occurred in a concentration dependent manner after 24 h and 48 h of coincubation. The study further confirmed the anti-microbial activity of Aronia melanocarpa leaf extract against Gram-negative bacterial species such as S. enterica, given that the bacterial growth could be decreased by approximately 65.5 ± 9.6% within 48 h post incubation at an extract concentration of 10% if compared to the control sample without any extract [19]. Interestingly, the same extract also exerted enhanced activity against the E. coli ATCC 10536 strain with a decrease of about 93% after a 48-h incubation period. Under the same conditions, the leaf extracts of Aronia melanocarpa were able to reduce the growth of the tested Gram-negative bacteria Acinetobacter baumannii and Pseudomonas fragi by approximately 93% and 98%, respectively, whereas growth of the Gram-positive B. thermosphacta could be decreased by 87%, and in case of S. aureus only by approximately 32%. Furthermore, scanning electron microscopic examinations revealed that the S. aureus cells showed pores resulting in destruction of the bacterial cell wall and subsequent cell death. Although the assessed morphological changes could not be observed for the E. coli bacteria, there was a significant decrease of the outer membrane permeability detected in all tested Gram-negative bacteria, including E. coli. Furthermore, Aronia melanocarpa leaf extract was able to decrease the E. coli DNA gyrase activity. Given that the E. coli DNA gyrase usually contributes to ensure the structural integrity of the DNA molecule by supercoiling the DNA, the authors hypothesized that this might lead to bacterial cell death upon inhibition [19].

A good host for Aronia fruit extract is presented by silica-type matrices, which have a large surface area, voluminous pores, a good capacity for external molecules and yet remain stable towards mechanical and thermal stress [20]. It was observed that the black chokeberry extract embedded in a silica-type matrix preserved its anti-bacterial as well as anti-oxidant activities. Interestingly, the Aronia-embedded matrix had an anti-microbial effect on the tested Gram-positive bacterial strains, whereas the Gram-negative bacteria remained unaffected. The most prominent anti-bacterial effect could be determined against the Streptococcus pyogenes ATCC 19615 strain with a MIC of 1 mg mL⁻¹, followed by the Streptococcus pneumoniae ATCC 49619 strain as well as the S. aureus ATCC 25923 strain both with a MIC and minimum bactericidal concentration (MBC) of 2 mg mL⁻¹. A slightly improved effect could be observed when the extract was encapsulated into a silica-type matrix, enriched with zinc oxide nanoparticles, compared to the free extract, which the authors related to a zinc oxide associated synergistic effect [20].

3.2 Studies on the application of Aronia melanocarpa in food packaging

Given the pronounced anti-bacterial activity of Aronia melanocarpa extracts directed against meat spoilage pathogens [18, 19], further studies addressed how to transfer this knowledge into practical application in order to prevent food products from spoilage and thus, to enhance food safety. Oun et al. [21] tested the anti-microbial activity of Aronia extract in a polyvinyl alcohol/chitosan-based (PVA/CS) composite film in vitro. This PVA/CS-Aronia film showed high anti-microbial activity against E. coli, given that the bacteria could be entirely eradicated after 9 h of incubation, which was also the case when tested against L. monocytogenes within a 6-h incubation period [21].

Furthermore, Lee et al. [22] tested the application of Aronia juice processing by-products for bioelastomer fabrication in food packaging. Therefore, polydimethylsiloxane (PDMS) was used for the fabrication of the bioelastomer, mixed with either 50% or 25% by-products from Aronia juice processing for the verum group and whole fruit freeze-dried extract in the control group. The PDMS polymers had been obtained from cocoa shell waste by-products and could offer a natural and sustainable alternative to conventional petrochemical-based films. When 50% Aronia juice by-product powder was used, the mechanical condition of the bioelastomer became crude and unstable and was therefore not suitable for use. Anti-microbial tests revealed that the bioelastomer exerted growth-inhibiting activities directed against Gram-positive bacteria such as S. aureus, but not against Gram-negative species including E. coli [22]. Whereas the anti-oxidant effects of the bioelastomer was approximately 1.4-times lower if compared to the control group, its anti-bacterial effect was about 1.5-fold higher [22].

3.3 In vivo studies in mice and men

The in vitro efficacy of black chokeberry against L. monocytogenes has already been described [12]. In order to investigate its potential in vivo activity, Gajic et al. [23] studied the effect of chokeberry fruit extract against L. monocytogenes in mice. Therefore, mice from the verum group were orally challenged with chokeberry extract (50 mg per kg body weight) for 7 days and infected with L. monocytogenes on day 8 by orogastric gavage, while the control group received vehicle instead. After the L. monocytogenes infection, the control mice showed a significant weight loss compared to the verum group that did not exhibit any decrease of body weight if compared to the initial state. After 48 h of infection, the L. monocytogenes burden in the spleen was measured by enumerating CFUs and revealed that the mice pre-treated with chokeberry extract displayed significantly lower splenic bacterial numbers than control mice. In addition, higher ratios of activated macrophages to cytotoxic T cells could be detected in the spleen as well as the gut-associated lymphoid tissue (GALT) of pre-treated versus control mice that were accompanied by more abundant ROS producing cells in Peyer's patches isolated from the former versus the latter. Hence, prophylactic chokeberry fruit extract application activated gut-associated myeloid cell function and resulted in enhanced clearance of L. monocytogenes in infected mice [23].

Notably, studies investigating the anti-bacterial activity of Aronia melanocarpa in human trials are very limited. Lee et al. [24] studied the anti-biofilm-forming effects of Aronia juice directed against Streptococcus mutans and Streptococcus sobrinus that comprise bacterial strains of the oral cavity known to be involved in dental caries development. Performing an in vitro biofilm formation assay first, the results showed that after 1 min of exposure to the 1:10-diluted Aronia juice, the streptococcal biofilm formation could be reduced by approximately 25–30%. Moreover, even pre-formed streptococcal biofilm could be reduced by up to 72%, whereas a decrease of streptococcal CFUs could not be observed under the same experimental conditions suggesting that the Aronia juice did not affect streptococcal growth. Interestingly, the oral rinsing with diluted Aronia juice led to a significant decrease of streptococcal numbers in the saliva if compared to the individuals from the control group that gargled with tap water. Furthermore, the authors showed that Aronia juice could counteract biofilm formation by decomposing extracellular RNA given that the experimental Aronia juice sample could completely degrade streptococcal RNA. In summary, the diluted Aroni juice did not affect the S. mutans and S. sobrinus growth in vitro but showed a significant decrease of streptococcal CFUs after oral rinsing in humans in vivo, whereas it could exert potent bacterial RNA-degrading activity [24].

Given that urinary tract infections (UTIs) are among the most prevalent bacterial infections in primary health care, Handeland et al. [25] tested the impact of black chokeberry juice consumption on the incidences of UTIs among residents of 6 nursing homes in Norway. Therefore, a 6-month crossover intervention study was performed, in which participants were enrolled into two groups (i.e., group A (n = 110) and group B (n = 126)) and offered a daily intake of black chokeberry juice between 90 and 160 mL. Group A received a placebo for the first three months followed by black chokeberry juice for the next period of three months and vice versa in group B. Medical treatments of infections during the study periods were documented by the nurses and revealed that each participant received an average of 1.51 antibiotic treatments, of which 55% were UTI indicated. There was no statistically significant difference in the number of infections treated during the intervention period compared to the control period before the treatment. Whereas in group A there was a 55% decrease in antibiotic treated UTIs in the post-treatment period compared to the intervention period, in group B, a decrease of 38% UTIs in the period after treatment could be observed. There was no effect noticeable on infectious diseases other than UTIs. According to the authors, the results underscore a long-term impact on the prevention of UTIs after consuming black chokeberry juice, which was detected for the first three months following the treatment period but was not evident thereafter [25].