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Key message Sustainability and safety aspects of plant cell cultures as food are presented. Applicability of dairy side streams as carbon source and use of natural growth enhancers in cultivation are shown. Abstract Biotechnologically produced cellular products are currently emerging to replace and add into the portfolio of agriculturally derived commodities. Plant cell cultures used for food could supplement current food production. However, still many aspects need to be resolved before this new food concept can enter the market. Issues related to sustainability and safety for human consumption are relevant for both consumers and regulators. In this study, two plant cell cultures, deriving from arctic bramble (Rubus arcticus) and birch (Betula pendula), were cultivated using lactose-rich dairy side streams as alternative carbon sources to replace sucrose. Biomasses were comparable to those of original plant cell culture media when up to 83% and 75% of the original sucrose was replaced by these side streams for arctic bramble and birch cell cultures, respectively. Furthermore, nutritional composition or sensory properties were not compromised. Synthetic plant growth regulators were replaced by natural components, such as coconut water and IAA for several subculture cycles. Finally, it was shown that only trace amounts of free growth regulators are present in the cells at the harvesting point and assessment by freshwater crustaceans assay indicated that toxicity of the cells was not exceeding that of traditionally consumed bilberry fruit.

Plant cell cultures from cloudberry (CL), lingonberry (LI), stone berry (ST), arctic bramble (AB), and strawberry (SB) were studied in terms of their polyphenol and carotenoid composition, antioxidant activity, antihemolytic activity and cytotoxicity effects on cancerous cells. High-resolution mass spectrometry data showed that LI, presented the highest antioxidant activity, contained the highest contents of flavones, phenolic acids, lignans, and total carotenoids, while CL, ST and SB presented the opposite behavior. AB and SB presented the lowest FRAP and CUPRAC values, while AB and CL presented the lowest reducing power. SB presented the lowest antioxidant activity measured by single electron transfer assays and the lowest content of lignans, phenolic acids, and flavones. CL and LI decreased the viability of in vitro mammary gland adenocarcinoma while only LI decreased the viability of in vitro lung carcinoma and showed protective effects of human erythrocytes against mechanical hemolysis.

Staphylococcus aureus is the most common cause of surgical site infections and its treatment is challenging due to the emergence of multi-drug resistant strains such as methicillin-resistant S. aureus (MRSA). Natural berry-derived compounds have shown antimicrobial potential, e.g., ellagitannins such as sanguiin H-6 and lambertianin C, the main phenolic compounds in Rubus seeds, have shown antimicrobial activity. The aim of this study was to evaluate the effect of sanguiin H-6 and lambertianin C fractionated from cloudberry seeds, on the MRSA growth, and as treatment of a MRSA biofilm development in different growth media in vitro and in vivo by using a murine wound infection model where sanguiin H-6 and lambertianin C were used to prevent the MRSA infection. Sanguiin H-6 and lambertianin C inhibited the in vitro biofilm development and growth of MRSA. Furthermore, sanguiin H-6 showed significant anti-MRSA effect in the in vivo wound model. Our study shows the possible use of sanguiin H-6 as a preventive measure in surgical sites to avoid postoperative infections, whilst lambertianin C showed no anti-MRSA activity.

We have developed an organic solvent-free process to enrich natural antimicrobials from the important Nordic Rubus berry species, especially from cloudberry. The process utilizes industrial berry byproducts as raw-material, and it is based on seed sanding technology and water-based extraction. The extracts showed strong antimicrobial activity against Gram-positive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). A concentration of 5 mg/mL totally eliminated these bacteria in 24 h. Effects against Gram-negative bacteria were modest. Polyphenol analysis of the hydrothermal extracts showed that the antimicrobial activity is correlated to ellagitannins, mainly dimeric sanguiin H-6 and sanguiin H-10 isomers. Sanguiin H-10 is not commonly found in intact cloudberry seeds, but it is formed from the dimeric and trimeric ellagitannins, during the extraction. Surprisingly hydrothermal extracts had no or minor effects on beneficial bacteria, Lactobacillus rhamnosus. This interesting finding might offer an application potential in controlling skin microbial pathogens, for example, in wound healing.

Main conclusion: The 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.

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.

Cosmetics are moving towards pharma and cosmeceuticals are entering the market. Modern biotechnology offers many advantages to develop new type of ingredients for industrial applications in a sustainable way. Due to the arctic growth conditions Nordic wild berries are rich sources of natural biomolecules with multifunctional effects to the skin. We have shown that phenolic compounds in berries very efficiently inhibit the growth of many human pathogens including skin pathogens, such as Staphylococcus aureus, S. epidermis and Pseudomonas aeruginosa without effecting the growth of beneficial bacteria. Several mechanisms of action, including the weakening of the outer membrane of Gram- negative bacteria, are involved in the growth inhibition. More interestingly, various berry phenolics are shown to specifically block cell-to-cell signalling (quorum sensing) in a bacterial community. Thus these phytochemicals can act in cosmetic preparations in dual way, as natural preservatives to prevent spoilage of the product, and as stabilizers of healthy skin microbiota by inhibiting the growth of harmful skin microbes and by supporting the growth of beneficial microbes.Bioprocessing including enzyme treatment and fermentation combined with special dry fractionation technologies of the berry material has resulted in increased bioactivities. Using plant cell and tissue culture technology whole biosynthetic capacity of the plant can be advantaged and even totally new beneficial compounds for the skin can be produced. The unusual phenolic profile of the cultured berry cells, as well as their fatty acid composition with a high proportion of α-linolenic acid and high protein content makes them a unique and interesting alternative for cosmetic industry. Efficient utilization of the biomolecules requires controlled delivery systems into the skin, such as microcurrent patches developed at VTT, which are based on renewable enzyme catalysts and renewable materials. The possibilities using natural plant cell-derived ingredients combined with new delivery system will also be discussed.

Tannins are a unique group of phenolic metabolites with molecular weights between 500 and 30000 Da, which are widely distributed in almost all plant foods and beverages. Proanthocyanidins and hydrolysable tannins are the two major groups of these bioactive compounds, but complex tannins containing structural elements of both groups and specific tannins in marine brown algae have also been described. Most literature data on food tannins refer only to oligomeric compounds that are extracted with aqueous-organic solvents, but a significant number of non-extractable tannins are usually not mentioned in the literature. The biological effects of tannins usually depend on their grade of polymerisation and solubility. Highly polymerised tannins exhibit low bioaccessibility in the small intestine and low fermentability by colonic microflora. This review summarises a new approach to analysis of extractable and non-extractable tannins, major food sources, and effects of storage and processing on tannin content and bioavailability. Biological properties such as antioxidant, antimicrobial and antiviral effects are also described. In addition, the role of tannins in diabetes mellitus has been discussed. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Gut is the site of active fermentation of non-digestible dietary components (dietary fibre and prebiotics) as well as bioconversion and absorption of plant-derived phenolics. These compounds have an important role in gut fermentation by influencing the composition of microflora and fermentation metabolites, and consequently by contributing to both local and systemic effects in humans. Possibilities to enhance viability and promote growth of probiotic bacteria by non-digestible food components have been a subject to extensive scientific interest in the last ten years. Gut bacteria are known to degrade and ferment dietary fibre, producing metabolites, especially short-chain fatty acids. They also mediate a number of important consequences through their further metabolism in the liver. Current research is at quick steps increasing our understanding about the interactions between gut microbes and bioactive dietary phenolics. Absorption and metabolism of phenolic compounds occurs along the digestive tract. Those compounds not absorbed or converted earlier enter the colon, and may be converted to metabolites concomitantly with carbohydrate fermentation. All the colonic metabolites can have effects on the epithelium at the site of conversion, and also affect the colonic flora locally. When absorbed the metabolites are found in plasma and urine and can have systemic health effects. The health effects of phenolic compounds have been studied extensively, but those of the metabolites are poorly known. As strong antimicrobial agents the phenolics might also have unpredictable effects on the composition of the intestinal flora.

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.

R. PUUPPONEN-PIMIA¨ , L. NOHYNEK, S. HARTMANN-SCHMIDLIN, M. KAHKONEN, M. HEINONEN, K. MAATTA¨ -RIIHINEN AND K.-M. OKSMAN-CALDENTEY. 2005. Aims: To investigate the effects of berries and berry phenolics on pathogenic intestinal bacteria and to identify single phenolic compounds being responsible for antimicrobial activity. Methods and Results: Antimicrobial activity of eight Nordic berries and their phenolic extracts and purified phenolic fractions were measured against eight selected human pathogens. Pathogenic bacterial strains, both Gram- positive and Gram-negative, were selectively inhibited by bioactive berry compounds. Cloudberry and raspberry were the best inhibitors, and Staphylococcus and Salmonella the most sensitive bacteria. Phenolic compounds, especially ellagitannins, were strong inhibitory compounds against Staphylococcus bacteria. Salmonella bacteria were only partly inhibited by the berry phenolics, and most of the inhibition seemed to originate from other compounds, such as organic acids. Listeria strains were not affected by berry compounds, with the exception of cranberry. Phenolic compounds affect the bacteria in different mechanisms. Conclusions: Berries and their phenolics selectively inhibit the growth of human pathogenic bacteria. Significance and Impact of the Study: Antimicrobial properties of berries could be utilized in functional foods. Furthermore these compounds would be of high interest for further evaluation of their properties as natural antimicrobial agents for food and pharmaceutical industry.

Aims: To investigate the antimicrobial properties of phenolic compounds present in Finnish berries against probiotic bacteria and other intestinal bacteria, including pathogenic species.Methods and Results: Antimicrobial activity of pure phenolic compounds representing flavonoids and phenolic acids, and eight extracts from common Finnish berries, was measured against selected Gram‐positive and Gram‐negative bacterial species, including probiotic bacteria and the intestinal pathogen Salmonella. Antimicrobial activity was screened by an agar diffusion method and bacterial growth was measured in liquid culture as a more accurate assay. Myricetin inhibited the growth of all lactic acid bacteria derived from the human gastrointestinal tract flora but it did not affect the Salmonella strain. In general, berry extracts inhibited the growth of Gram‐negative but not Gram‐positive bacteria. These variations may reflect differences in cell surface structures between Gram‐negative and Gram‐positive bacteria. Cloudberry, raspberry and strawberry extracts were strong inhibitors of Salmonella. Sea buckthorn berry and blackcurrant showed the least activity against Gram‐negative bacteria.Conclusions: Different bacterial species exhibit different sensitivities towards phenolics.Significance and Impact of the Study: These properties can be utilized in functional food development and in food preservative purposes.

Scope: Ellagitannins are polyphenols abundant in strawberries, raspberries and cloudberries. The effects of a mixture of these berries were studied in subjects with features of metabolic syndrome. The study focused on cardiovascular risk factors, gut microbiota and ellagitannin metabolites. Methods and Results: Twenty subjects consumed 300 g of ellagitannin rich berries daily for 8 weeks ina randomized controlled trial. A control group, with twelve subjects, ate no berries. Most measured variables did not differ between the two study groups. However, minor positive alterations were observed in systolic blood pressure, LDL cholesterol, 8-isoprostane, TRAP and resistin. Statistical modelling of lipidprofiles pinpointed a group of 20 lipids for sample separation, indicating that they may be responsible for known positive health effects of berries. Berry supplementation caused changes to microbiota in 35% of the subjects. All these changes were related to either Families of Ruminococcaceae or Lachnospiraceae,which are the main butyrate-producing bacteria in the human colon. A majority of the subjects in the berry group were identified as urolithin producers.Conclusions: Ellagitannin rich berry supplementation may have positive effects on serum lipid profiles and on gut microbiota, which might modify bioavailability of the berry ellagitannins. Future studies in humans are warranted.

Berries are rich in phenolic compounds with beneficial biological properties for human health. Arctic bramble (Rubus arcticus) grows in borealic zone, and its ruby red berries are well known for good taste and flavour. In addition, arctic bramble is rich in bioactive phenolic compounds, especially the complex phenolic polymer ellagitannin, which is reported to have e.g. in vitro anti-oxidant activities and antimicrobial effects against human pathogens. The colour of arctic bramble originates from anthocyanins, which also possess variable beneficial effects on human health. The crop of both wild and cultivated arctic bramble is very low, and therefore cell cultures are the potential choice for production of already characterized and novel secondary metabolites for food and pharmaceutical purposes. Arctic bramble cell cultures were established from cuts of sterile in vitro leaves on medium with plant hormones kinetin and NAA (alpha-Naphtalen-acetic acid). Callus lines were grown on the hormone medium, and good growth and bright colours were used for selection criteria for maintenance and initiation of suspension cultures. Phenolic compounds were measured from stable callus and suspension cultures obtained, and selected culture was used for elicitation experiment aiming to increase production of phenolic secondary metabolites. Elicitors used were methyl jasmonate, ethephon, S-ABA and chitosan, and they were introduced in the cultures in late logarithmic growth phase. Samples collected at different time points were filtered, and the cell mass was freeze-dried and extracted with methanol. Freeze-dried extracts were analysed by HPLC-DAD. In callus and suspension cultures phenolic acids were the main phenolics detected in methanol extracts. Elicitation clearly enhanced the production of some phenolic compounds in suspension cultures, and elicitation with S-ABA also promoted synthesis of a new phenolic compound, which still needs to be characterized.