Natural Carotenoids: Recent Advances on Separation from Microbial Biomass and Methods of Analysis

Papapostolou H, Kachrimanidou V, Alexandri M, Plessas S, Papadaki A, Kopsahelis N.

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Biotechnologically produced carotenoids occupy an important place in the scientific research. Owing to their role as natural pigments and their high antioxidant properties, microbial carotenoids have been proposed as alternatives to their synthetic counterparts. To this end, many studies are focusing on their efficient and sustainable production from renewable substrates. Besides the development of an efficient upstream process, their separation and purification as well as their analysis from the microbial biomass confers another important aspect. Currently, the use of organic solvents constitutes the main extraction process; however, environmental concerns along with potential toxicity towards human health necessitate the employment of “greener” techniques. Hence, many research groups are focusing on applying emerging technologies such as ultrasounds, microwaves, ionic liquids or eutectic solvents for the separation of carotenoids from microbial cells. This review aims to summarize the progress on both the biotechnological production of carotenoids and the methods for their effective extraction. In the framework of circular economy and sustainability, the focus is given on green recovery methods targeting high-value applications such as novel functional foods and pharmaceuticals. Finally, methods for carotenoids identification and quantification are also discussed in order to create a roadmap for successful carotenoids analysis.

Antioxidants 2023, 12(5), 1030.

Upgrading major waste streams derived from the biodiesel industry and olive mills via microbial bioprocessing with non-conventional Yarrowia lipolytica strains 

Sarris D, Tsouko E, Kothri M, Anagnostou M, Karageorgiou E, Papanikolaou S.

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This study reports the development of a bioprocess involving the valorization of biodiesel-derived glycerol as the main carbon source for cell proliferation of Yarrowia lipolytica strains and production of metabolic compounds, i.e., citric acid (Cit), polyols, and other bio-metabolites, the substitution of process tap water with olive mill wastewater (OMW) in batch fermentations, and partial detoxification of OMW (up to 31.1% decolorization). Increasing initial phenolics (Phen) of OMW-glycerol blends led to substantial Cit secretion. Maximum Cit values, varying between 64.1–65.1 g/L, combined with high yield (YCit/S = 0.682–0.690 g Cit/g carbon sources) and productivity (0.335–0.344 g/L/h) were achieved in the presence of Phen = 3 g/L. The notable accumulation of endopolysaccharides (EPs) on the produced biomass was determined when Y. lipolytica LMBF Y-46 (51.9%) and ACA-YC 5033 (61.5%) were cultivated on glycerol-based media. Blending with various amounts of OMW negatively affected EPs and polyols biosynthesis. The ratio of mannitol:arabitol:erythritol was significantly affected (p < 0.05) by the fermentation media. Erythritol was the major polyol in the absence of OMW (53.5–62.32%), while blends of OMW-glycerol (with Phen = 1–3 g/L) promoted mannitol production (54.5–76.6%). Nitrogen-limited conditions did not favor the production of cellular lipids (up to 16.6%). This study addressed sustainable management and resource efficiency enabling the bioconversion of high-organic-load and toxic waste streams into valuable products within a circular bioeconomy approach.

Fermentation 20239(3), 251.

Development of Dairy Products Fortified with Plant Extracts: Antioxidant and Phenolic Content Characterization 

Kandyliari A, Potsaki P, Bousdouni P, Kaloteraki C, Christofilea M, Almpounioti K, Moutsou A, Fasoulis C, Polychronis L, Gkalpinos V, Tzakos A, Koutelidakis A.

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In recent decades, there has been growing interest in the fortification of dairy products with antioxidants and phenolics derived from plant byproducts and herbs. The present study focused on the analysis of dairy products, including kefir, cream cheese, yogurt, and vegan yogurt, enhanced with aqueous extracts of plant byproducts (Citrus aurantium peel, Citrus limon peel and Rosa canina seed) and herbs (Sideritis spp., Hypericum perforatumOriganum dictamnusMentha pulegium L., Melissa oficinallisMentha spicata L. and Lavandula angustifolia) to characterize their antioxidant content, phenolic profile, and organoleptic characteristics. Antioxidant and phenolic content were determined by Folin–Ciocalteu and ferric reducing antioxidant power (FRAP) assays and presented values up to 46.61 ± 7.22 mmol Fe2+/L and 82.97 ± 4.29 mg gallic acid (GAE)/g, respectively for the aqueous extracts, as well as up to 0.68 ± 0.06 mmol Fe2+/L and 2.82 ± 0.36 mg GAE/g for the fortified dairy products. The bioavailability of antioxidants and phenolics in fortified foods was determined after in vitro digestion and ranged between 4 and 68%. The phytochemical profile of the aqueous extracts was determined by mass spectrometry, and 162 phytochemicals were determined, from which 128 belong to the polyphenol family including flavonoids and phenolic acids. Furthermore, most of the identified compounds have been recorded to possess enhanced antioxidant capacity in correlation to the in vitro findings. Finally, organoleptic evaluation showed an overall acceptability around 3.0 ± 1.0 on a 5-point scale. In conclusion, the studied plants and herbal extracts can be used for the fortification of a variety of dairy products with potential positive effects on human health.

Antioxidants 202312(2), 500.

Exploring the potential of novel R. kratochvilovae red yeasts towards the sustainable synthesis of natural carotenoid

Sereti F,  Papadaki A, Alexandri M, Kachrimanidou V, Kopsahelis N.

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The development of microbial cell factories is nowadays a main route for the production of safe and environmentally friendly bioactive compounds. Rhodosporidium kratochvilovae yeasts constitute an unexplored source for the production of natural carotenoids, thus further insights on their metabolism and fermentative behavior would contribute towards the sustainable synthesis of bioactive compounds. The aim of this study was to investigate the ability of two R. kratochvilovae isolates (FMCC Y-42 and Y-43) to produce carotenoids under different experimental conditions. Cell growth, carotenoid synthesis and lipid accumulation were evaluated using synthetic media containing either glucose or mixtures of glucose:galactose (1:1), under different carbon-to-nitrogen (C/N) ratios. Carotenogenesis was highly affected by C/N ratio in both strains, while a competitive trend with lipid accumulation was also observed. Yeast strain Y-42 reached the highest concentration of total carotenoids (2.59 mg/L) when grown on glucose-based medium at a C/N ratio of 80 after 39 h of fermentation. Likewise, the strain Y-43 produced approximately 2 mg/L of total carotenoids from both glucose (C/N of 80) and its mixture with galactose (C/N 80 and 120), with similar biomass concentration (9.3, 10.3 and 10.9 g/L, respectively). HPLC-DAD analysis of carotenoid extracts indicated that the main carotenoid was β-carotene (60–75%) followed by γ-carotene (9–18%), torulene (6–13%) and torularhodin (5–17%), regardless the fermentation conditions or the strain-employed. This is the first study to reveal γ-carotene synthesis by R. kratochvilovae strains. The obtained results provided new knowledge on the production of natural carotenoids by novel red yeast strains and could pave the way towards their application in sustainable functional food products and nutraceuticals.

Sustainable Chemistry and Pharmacy 2023, 31, 2352.


Gregoria Mitropoulou, Ioanna Prapa, Anastasios Nikolaou, Konstantinos Tegopoulos, Theodora Tsirka, Nikos Chorianopoulos, Chrysoula Tassou, Petros Kolovos, Maria E. Grigoriou, Yiannis Kourkoutas

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Background: Cheese microbiome plays a key role in determining the organoleptic and physico-chemical properties and may be also used as an authenticity tool for distinguishing probiotic cultures. Due to significant reduction of cell viability often witnessed during food production processes and storage, immobilization is proposed to ascertain high probiotic cell loads required to confer the potential health benefits. Hence, the aim of the present study was to investigate the effect of free or immobilized Lactiplantibacillus plantarum T571 on whey protein on feta cheese microbiome. Methods: Next-Generation Sequencing technology was used to investigate cheese microbiome. Cheese samples containing free or immobilized Lactiplantibacillus plantarum T571 (a wild type strain isolated from Feta cheese brine) on whey protein, along with products containing commercial starter culture, were analyzed. Results: The results showed a great diversity of bacteria and fungi genera among the samples. An increased presence of Lactobacillus OTUs in cheese with immobilized cells on whey protein was witnessed, highlighting the survival of the strain in the final product. The immobilized culture had also a significant impact on other genera, such as LactococcusLeuconostoc and Debaryomyces, which are associated with improved technological characteristics and health benefits. Conclusions: Enrichment of feta cheese with immobilized potential probiotics to secure cell viability consists of an industrial challenge and leads to distinct microbiome composition that may be used as a valuable food authenticity tool.

Front. Biosci. (Elite Ed). 2022, 14(4), 31.

Sustainable Food Systems: The Case of Functional Compounds towards the Development of Clean Label Food Products

Alexandri M, Kachrimanidou V, Papapostolou H, Papadaki A, Kopsahelis N.

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The addition of natural components with functional properties in novel food formulations confers one of the main challenges that the modern food industry is called to face. New EU directives and the global turn to circular economy models are also pressing the agro-industrial sector to adopt cradle-to-cradle approaches for their by-products and waste streams. This review aims to present the concept of “sustainable functional compounds”, emphasizing on some main bioactive compounds that could be recovered or biotechnologically produced from renewable resources. Herein, and in view of their efficient and “greener” production and extraction, emerging technologies, together with their possible advantages or drawbacks, are presented and discussed. Modern examples of novel, clean label food products that are composed of sustainable functional compounds are summarized. Finally, some action plans towards the establishment of sustainable food systems are suggested.

Foods 2022, 11(18), 1796.

Probiotics and Phytochemicals: Role on Gut Microbiota and Efficacy on Irritable Bowel Syndrome, Functional Dyspepsia, and Functional Constipation

Bousdouni, P., Kandyliari, A., & Koutelidakis, A.

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Functional gastrointestinal disorders (FGIDs), such as irritable bowel syndrome, functional constipation, and functional dyspepsia, have had a high prevalence over the past few years. Recent evidence suggests that functional foods and bioactive compounds, such as probiotics and phytochemicals, may have a positive effect in treating the symptoms of the above diseases. In this systematic review study, 32 published studies were selected with the use of comprehensive scientific databases, according to PRISMA guidelines, with emphasis on recent interventional studies that reflect the effect of probiotics and selected phytochemicals on the improvement of FGID symptoms. The bioactive compounds in the selected studies were administered to patients either in capsule form or in enriched food products (yogurt, juice, etc.). According to the results, there is a correlation between the consumption of probiotics and phytochemicals, such as polyphenols, and the relief of symptoms in selected gastrointestinal disorders. Enriching foods that are regularly consumed by the population, such as fruit juices, yogurt, and cheese, with ingredients that may have a positive effect on gastrointestinal disorders, could be a possible novel goal for the management of these diseases. However, further evidence is required for the role of probiotics and phytochemicals in FGIDs to be fully understood.

Gastrointest. Disord. 20224(1), 30.

Development of a DHA-Losartan hybrid as a potent inhibitor of multiple pathway-induced platelet aggregation

Tsiailanis, A. D., Vrettos, E. I., Choleva, M., Kiriakidi, S., Ganai, A. M., Patha, T. K., Karpoormath R., Mavromoustakos T., Fragopoulou E., & Tzakos, A. G. 

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Despite the scientific progression in the prevention and treatment of cardiovascular diseases (CVDs) they remain the leading cause of mortality and disability worldwide. The classic treatment involves the simultaneous dosing of two antiplatelet drugs, aspirin and clopidogrel/prasugrel. However, besides drug resistance, severe side effects have been also manifested including acute bleeding and toxicity. Thus, new therapeutic agents with enhanced efficacy and diminished side effects are of importance. Towards this end, omega-3 (ω-3) fatty acids have demonstrated potent efficacy against CVDs through inhibiting platelet aggregation that bears a pivotal role in atherothrombosis. Another factor that displays a critical role in the pathogenesis of cardiovascular diseases is the renin-angiotensin system (RAS), and especially the AT1R blocker losartan that has been reported to exert antiplatelet activity mediated by this receptor. Along these lines, we envisaged developing a molecular hybrid consisted of docosahexaenoic acid (ω-3 fatty acid) and losartan, that could exert a notable antiplatelet effect against CVDs. The design and synthesis of the new DHA-losartan hybrid, designated DHA-L, bestowed with the additive properties of the parent compounds, is reported. In silico studies were first exploited to validate the potential of DHA-L to retain losartan’s ability to bind AT1R. The antiplatelet activity of DHA-L was evaluated against in vitro platelet aggregation induced by several platelet agonists. Notably, the hybrid illustrated a pleiotropic antiplatelet profile inhibiting platelet aggregation through multiple platelet activation pathways including P2Y12, PAR-1 (Protease-Activated Receptor-1), PAF (Platelet Activating Factor), COX-1 (cyclooxygenase-1) and collagen receptors. The stability of DHA-L in human plasma and in a wide range of pH values was also evaluated over time using an HPLC protocol. The hybridization approach described herein could pave the way for the development of novel potent multitargeted therapeutics with enhanced antiplatelet profile.

Journal of Biomolecular Structure and Dynamics 2021, 1-12

Total Antioxidant Capacity and Phenolic Content of 17 Mediterranean Functional Herbs and Wild Green Extracts from North Aegean, Greece

Kaloteraki, C., Almpounioti, K., Potsaki, P., Bousdouni, P., Kandyliari, A. & Koutelidakis, A.

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Functional foods are considered beneficial to human health due to their high nutritional values. Currently, there is development in research to identify natural functional food sources high in bioactive compounds, such as antioxidants and polyphenols, as they are shown to contribute towards decreasing oxidative stress and maintaining well-being in human health. The objective of the present study was to determine the total antioxidant capacity and total phenolic content of Mediterranean medicinal natural herbs and wild greens, aiming to create a database of medicinal plants for future potential use as functional food ingredients. A total of seventeen selected herbs and wild greens from Lemnos Island (Greece) were purchased between June and July of 2021. Extracts were prepared with an ultrasound water bath at 70 °C for 60 min. The antioxidant capacity was measured with ferric reducing antioxidant power (FRAP) assay and the total polyphenolic content was determined with the Folin–Ciocalteu reagent method. The mean phenolic values ranged from 0.18 to 2282.80 mg of gallic acid/g of sample, whereas Hypericum perfoliatum L. and Hypericum perforatum L., Salvia spp., and Sideritis sp. displayed the highest contents (p < 0.05). Melissa officinalis L., Mentha Spicata, and Thymbra capitata L. obtained the highest total antioxidant capacity values greater than 12.20 mmol/L, with an average range of 0.06 to 30.58 mmol of Fe2+/L of extract (p < 0.05). Pistacia lentiscus var. chiaCrataegus azarolus L., and Pancratium Maritimum obtained the lowest contents from both methods. Studies have shown that medicinal plants with total antioxidant values higher than 0.87 mmol/L could be applicable in the fortification of food products (i.e., yogurt). Therefore, this study suggests that most of the above medicinal plants could be a potential source of antioxidants for functional food applications while their use can enhance Mediterranean diet principles.

Biol. Life Sci. Forum 20216(1), 43.

High-Temperature Semi-Dry and Sweet Low Alcohol Wine-Making Using Immobilized Kefir Culture

Nikolaou, N. & Kourkoutas, Y.

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Low alcohol wines (≤10.5% vol) represent novel wine products steadily gaining the commercial market interest. Considering the technological advancements of immobilized systems in association with the drastic reduction of industrial operational costs in high-temperature wine-making in regions with tropical climate or hot summer periods, the aim of the present study was to assess the fermentation efficiency of both wet and freeze-dried immobilized kefir culture on natural supports in low alcohol wine production at high temperatures (>30 °C). Immobilized kefir culture was evaluated and compared to free cells in repeated batch fermentations for 3 months, indicating high operational stability, and found suitable for simultaneous alcoholic and malolactic low alcohol wine fermentation at temperatures up to 45 °C. High ethanol productivity [up to 55.3 g/(Ld)] and malic acid conversion rates (up to 71.6%), which could be adopted by the industrial sector, were recorded. Principal Component Analysis (PCA) revealed that the state of the cells rather than the nature of kefir culture affected significantly the content of minor volatiles determined by Head Space Solid-Phase Microextraction (HS-SPME) Gas Chromatography–Mass Spectrometry (GC/MS) analysis. Notably, all new products were of high quality and approved by the sensory panel. The results suggested a high industrial potential of the proposed technology in semi-dry low alcohol wine-making at 37 °C and in developing novel wine products with a sweet (liquoreux) character at 45 °C.

Fermentation 2021, 7(2), 45.