Liisa Nohynek

Berry Phenolics: Antimicrobial Properties and Mechanisms of Action Against Severe Human Pathogens

Abstract: Antimicrobial activity and mechanisms of phenolic extracts of 12 Nordic berries were studied against selected human pathogenic microbes. The most sensitive bacteria on berry phenolics were Helicobacter pylori and Bacillus cereus. Campylobacter jejuni and Candida albicans were inhibited only with phenolic extracts of cloudberry, raspberry, and strawberry, which all were rich in ellagitannins. Cloudberry extract gave strong microbicidic effects on the basis of plate count with all studied strains. However, fluorescence staining of liquid cultures of virulent Salmonella showed viable cells not detectable by plate count adhering to cloudberry extract, whereas Staphylococcus aureus cells adhered to berry extracts were dead on the basis of their fluorescence and plate count. Phenolic extracts of cloudberry and raspberry disintegrated the outer membrane of examined Salmonella strains as indicated by 1-N-phenylnaphthylamine (NPN) uptake increase and analysis of liberation of [14C]galactose- lipopolysaccharide. Gallic acid effectively permeabilized the tested Salmonella strains, and significant increase in the NPN uptake was recorded. The stability of berry phenolics and their antimicrobial activity in berries stored frozen for a year were examined using Escherichia coli and nonvirulent Salmonella enterica sv. Typhimurium. The amount of phenolic compounds decreased in all berries, but their antimicrobial activity was not influenced accordingly. Cloudberry, in particular, showed constantly strong antimicrobial activity during the storage.

Bioactive berry compounds—novel tools against human pathogens

Berry fruits are rich sources of bioactive compounds, such as phenolics and organic acids, which have antimicrobial activities against human pathogens. Among different berries and berry phenolics, cranberry, cloudberry, raspberry, strawberry and bilberry especially possess clear antimicrobial effects against, e.g. Salmonella and Staphylococcus. Complex phenolic polymers, like ellagitannins, are strong antibacterial agents present in cloudberry and raspberry. Several mechanisms of action in the growth inhibition of bacteria are involved, such as destabilisation of cytoplasmic membrane, permeabilisation of plasma membrane, inhibition of extracellular microbial enzymes, direct actions on microbial metabolism and deprivation of the substrates required for microbial growth. Antimicrobial activity of berries may also be related to antiadherence of bacteria to epithelial cells, which is a prerequisite for colonisation and infection of many pathogens. Antimicrobial berry compounds may have important applications in the future as natural antimicrobial agents for food industry as well as for medicine. Some of the novel approaches are discussed.

Lingonberry (Vaccinium vitis-idaea) and European Cranberry (Vaccinium microcarpon) Proanthocyanidins: Isolation, Identification, and Bioactivities

European, small-fruited cranberries (Vaccinium microcarpon) and lingonberries (Vaccinium vitis-idaea) were characterized for their phenolic compounds and tested for antioxidant, antimicrobial, antiadhesive, and antiinflammatory effects. The main phenolic compounds in both lingonberries and cranberries were proanthocyanidins comprising 63-71% of the total phenolic content, but anthocyanins, hydroxycinnamic acids, hydroxybenzoic acids, and flavonols were also found. Proanthocyanidins are polymeric phenolic compounds consisting mainly of catechin, epicatechin, gallocatechin, and epigallocatechin units. In the present study, proanthocyanidins were divided into three groups: dimers and trimers, oligomers (mDP 4-10), and polymers (mDP > 10). Catechin, epicatechin, A-type dimers and trimers were found to be the terminal units of isolated proanthocyanidin fractions. Inhibitions of lipid oxidation in liposomes were over 70% and in emulsions over 85%, and in most cases the oligomeric or polymeric fraction was the most effective. Polymeric proanthocyanidin extracts of lingonberries and cranberries were strongly antimicrobial against Staphylococcus aureus, whereas they had no effect on other bacterial strains such as Salmonella enterica sv. Typhimurium, Lactobacillus rhamnosus and Escherichia coli. Polymeric fraction of cranberries and oligomeric fractions of both lingonberries and cranberries showed an inhibitory effect on hemagglutination of E. coli, which expresses the M hemagglutin. Cranberry phenolic extract inhibited LPS-induced NO production in a dose-dependent manner, but it had no major effect on iNOS of COX-2 expression. At a concentration of 100 μg/mL cranberry phenolic extract inhibited LPS-induced IL-6, IL-1β and TNF-α production. Lingonberry phenolics had no significant effect on IL-1β production but inhibited IL-6 and TNF-α production at a concentration of 100 μg/mL similarly to cranberry phenolic extract. In conclusion the phenolics, notably proanthocyanidins (oligomers and polymers), in both lingonberries and cranberries exert multiple bioactivities that may be exploited in food development.

Enzyme-Assisted Processing Increases Antimicrobial and Antioxidant Activity of Bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.

Effects of ellagitannin‐rich berries on blood lipids, gut microbiota, and urolithin production in human subjects with symptoms of metabolic syndrome

Ellagitannins are polyphenols abundant in strawberries, raspberries, and cloudberries. The effects of a mixture of these berries were studied in a randomized controlled trial with subjects having symptoms of metabolic syndrome. The study focused on serum lipid profiles, gut microbiota, and ellagitannin metabolites. The results indicate that bioavailability of ellagitannins appears to be dependent on the composition of gut microbiota.

Rowanberry Phenolics: Compositional Analysis and Bioactivities

Berries contain a large variety of different phenolic compounds such as anthocyanins, flavonols, tannins, and phenolic acids. Due to variation in the nature and content of the phenolic compounds, the antioxidant effect and other bioactivities of berry phenolics are strongly dependent on the berry raw material as the activities differ between the different phenolic constituents. In the present study, wild rowanberries ( Sorbus aucuparia ) and four cultivated sweet rowanberries, Burka, Granatnaja, Titan, and Zoltaja, were characterized for their phenolic composition and screened for antioxidant, antimicrobial, and antiadhesive activities. The HPLC and LC-MS analyses of phenolic composition revealed that the main phenolic constituents were caffeoylquinic acids, varying from 56 to 80% total phenolics. The cultivated species contained less caffeoylquinic acids and more anthocyanins (up to 28.5%). The phenolics derived from wild rowanberries were significantly effective at inhibiting lipid oxidation both in liposomes and in emulsions, especially when assessed by inhibition of the formation of hexanal (86-97% inhibition depending on concentration). The increase in anthocyanin content in the cultivated species did not result in significantly increased antioxidant activity. Both wild and cultivated rowanberry phenolics exhibited a bacteriostatic effect toward Staphylococcus aureus . In addition, the phenolic extract from Zoltaja was weakly inhibitory toward Salmonella sv. Typhimurium, whereas both Zoltaja- and Granatnaja-derived phenolics retarded Escherichia coli growth. The phenolic extracts of wild rowanberries and Burka showed an inhibitory effect on hemagglutination of E. coli HB101 (pRR7), which expresses the M hemagglutinin. It can be concluded that cultivation of rowanberries resulted in increased anthocyanin content, but this did not diminish their bioactivity in comparison to wild rowanberries rich in caffeoylquinic acids.

Algae–bacteria association inferred by 16S rDNA similarity in established microalgae cultures

Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae‐associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga–Flavobacterium–Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities.

Cloudberry (Rubus chamaemorus) cell culture with bioactive substances: Establishment and mass propagation for industrial use

The availability and quality of wild berry material is strongly affected by seasonal variations, human resources, and both chemical and biological pollutants. Here we describe for the first time the industrial scale biotechnological production of cloudberry (Rubus chamaemorus) cells of consistent quality and defined chemical composition. Callus was initiated from sterile cuts of the wild cloudberry leaves. Bright yellow, soft callus of uniform quality was selected by regular subculturing, and a stable line successfully used for suspension culture was obtained within 18 months. The cultivation process of the cell culture was initiated in 250 mL shake flasks and stepwise expanded to a stirred tank bioreactor of 300 L working volume. Each growth period in flasks and fed‐batch cultures in bioreactors was 10 ± 3 days. Viability of the cultures was approximately 90% throughout the cultivation processes. The 300 L culture was harvested by pressure filtration and the biomass was freeze‐dried. The 300 L yields of fresh cells and freeze‐dried material were 19 and 2 kg, respectively. The unusual phenolic profile of cloudberry cells including flavanols, as well as their fatty acid composition with a high proportion of α‐linolenic acid and high protein content, make them a unique and interesting alternative for industrial applications for various industrial fields.

Variability in the production of tannins and other polyphenols in cell cultures of 12 Nordic plant species

AbstractMain conclusionThe polyphenol profiles of 18 cell cultures from 12 plant species were screened. The detected polyphenol fingerprints were diverse and differed from polyphenol profiles typically found in corresponding plant species. Cell cultures originating from 12 different plant species growing or grown in the Nordic countries were screened for their ability to synthesize polyphenols to assess their suitability for future studies and applications. The focus was on plant families Rosaceae and Ericaceae. On average, the Rosaceae cultures were the most efficient to produce hydrolysable tannins and the Ericaceae cultures were the most efficient to produce proanthocyanidins. This is in line with the general trend of polyphenols found in Rosaceae and Ericaceae leaves and fruits, even though several individual cell cultures differed from natural plants in their polyphenolic composition. Overall, several of the studied cell cultures exhibited capability in producing a large variety of polyphenols, including tannins with a high molecular weight, thus also showing promise for further studies concerning, for example, the accumulation of specific polyphenols or biosynthesis of polyphenols in the cell cultures.

Fermentation and dry fractionation increase bioactivity of cloudberry (Rubus chamaemorus)

Phenolic composition and bioactivity of cloudberry was modified by bioprocessing, and highly bioactive fractions were produced by dry fractionation of the press cake. During fermentation polymeric ellagitannins were partly degraded into ellagic acid derivatives. Phenolic compounds were differentially distributed in seed coarse and fine fractions after dry fractionation process. Tannins concentrated in fine fraction, and flavonol derivatives were mainly found in coarse fraction. Ellagic acid derivatives were equally distributed between the dry fractions. Fermentation and dry fractionation increased statistically significantly anti-adhesion and anti-inflammatory activity of cloudberry. The seed fine fraction showed significant inhibition of P fimbria-mediated haemagglutination assay of uropathogenic Escherichia coli. The seed coarse fraction significantly reduced NO and IL-6 production and iNOS expression in activated macrophages. Fermentation did not affect antimicrobial activity, but slight increase in activity was detected in dry fractions. The results indicate the potential of cloudberry in pharma or health food applications.