Novel Probiotic Candidates in Artisanal Feta-Type Kefalonian Cheese: Unveiling a Still-Undisclosed Biodiversity.

Autochthonous dairy lactic acid bacteria (LAB) isolates encompass a natural source of starter, adjunct, or probiotic candidates. In this context, traditionally manufactured, using exclusively animal rennet, Feta-type cheeses were collected from five farms located in different regions of Kefalonia island (Greece). The primary objective of this study was to isolate and characterize novel LAB, thereby exploring the unmapped microbial communities of Kefalonian Feta-type cheese and identifying new potential probiotics. The initial screening, included a preliminary gastrointestinal (GI) tolerance assessment (acidic conditions and bile salts), followed by their safety evaluation (hemolytic activity and antibiotic susceptibility). Based on the preliminary screening, selected strains underwent molecular identification and were further investigated for their probiotic attributes (lysozyme and phenol resistance, antimicrobial traits, antidiabetic aspects, cholesterol reduction and adhesion, adhesion to Caco-2 cells, and milk acidification potential). The results showed that 49, out of the 93 retrieved isolates, exhibited resistance to GI conditions, whereas 18 met the safety criteria. The molecular identification revealed strains belonging to the species Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and Lacticaseibacillus paracasei. The selected rod-shaped 14 isolates displayed a potential probiotic character. The best-performing isolates concerning cholesterol assimilation and adhesion, α-glucosidase inhibition, and epithelial adherence were Lpb. plantarum F89, F162, and F254 and Lcb. paracasei F214 and F216, whereas Lcb. paracasei F70 showed potential as a defined strain starter. The present study explores for the first time the biodiversity of traditionally fermented microbial communities in Kefalonian Feta-type cheese, revealing novel potential probiotic strains that can contribute to the development of innovative functional food products.

Carotenoids production by Rhodosporidium paludigenum yeasts: Characterization of chemical composition, antioxidant and antimicrobial properties.

The high market potential imposed by natural carotenoids has turned the scientific interest in search for new strains, capable of synthesizing a wide spectrum of these pigments. In this study, Rhodosporidium paludigenum NCYC 2663 and 2664 were investigated for carotenoids production and lipid accumulation utilizing different carbon sources (glucose, fructose, sucrose, mixture of glucose: galactose). Strain R. paludigenum 2663 produced the highest total carotenoids titer (2.21 mg/L) when cultivated on sucrose, together with 4 g/L lipids (30% w/w content) and 7 g/L exopolysaccharides. In the case of R. paludigenum 2664, glucose favored the production of 2.93 mg/L total carotenoids and 1.57 g/L lipids (31.8% w/w content). Analysis of the chemical profile during fermentation revealed that ß-carotene was the prominent carotenoid. Strain 2663 co-produced γ-carotene, torulene and torularhodin in lower amounts, whereas 2664 synthesized almost exclusively ß-carotene. The produced lipids from strain 2663 were rich in oleic acid, while the presence of linoleic acid was also detected in the lipoic fraction from strain 2664. The obtained carotenoid extracts exhibited antioxidant (IC50 0.14 mg/mL) and high antimicrobial activity, against common bacterial and fungal pathogenic strains. The results of this study are promising for the utilization of biotechnologically produced carotenoids in food applications.

Ganoderma lucidum Mycelia Mass and Bioactive Compounds Production through Grape Pomace and Cheese Whey Valorization.

Numerous compounds obtained from the medicinal mushroom Ganoderma lucidum have evidenced renowned bioactive characteristics. Controlled fermentation to generate fungal mycelia confers several advantages, specifically when the valorization of agro-industrial streams as fermentation feedstocks is included. Submerged fermentation of a newly isolated Greek strain of G. lucidum was performed using conventional synthetic media and, also, grape pomace extract (GPE) and cheese whey permeate (CWP) under static and shaking conditions. Under shaking conditions, maximum biomass with GPE and supplementation with organic nitrogen reached 17.8 g/L. The addition of an elicitor in CWP resulted in a significant improvement in biomass production that exceeded synthetic media. Overall, agitation demonstrated a positive impact on biomass productivity and, therefore, on process optimization. Crude intracellular and extracellular polysaccharides were extracted and evaluated regarding antioxidant activity and polysaccharide and protein content. FTIR analysis confirmed the preliminary chemical characterization of the crude extracts. This study introduces the design of a bioprocessing scenario to utilize food industry by-products as onset feedstocks for fungal bioconversions to obtain potential bioactive molecules within the concept of bioeconomy.

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

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.

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

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. Μodern 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.

Novel Probiotic/Bacterial Cellulose Biocatalyst for the Development of Functional Dairy Beverage.

The development of innovative functional products with potential health benefits, under the concept of bio-economy, is flourishing. This study undertook an evaluation of non-dairy lactobacilli Lactiplantibacillus pentosus B329 and Lactiplantibacillus plantarum 820 as "ready to use" starter cultures. Lactic acid bacteria (LAB) cultures were evaluated for their fermentation efficiency, before and after freeze-drying, using cheese whey (CW) as a fermentation substrate and subsequent immobilization on bacteria cellulose (BC) to produce a novel biocatalyst. The biocatalyst was applied in functional sour milk production and compared with free cells via the assessment of physicochemical and microbiological properties and sensory evaluation. Evidently, LAB strains exhibited high fermentative activity before and after freeze-drying. Results of a 5-month storage stability test showed that viability was 19% enhanced by immobilization on BC, supporting the concept of "ready to use" cultures for the production of fermented beverages. Likewise, sour milk produced by the BC biocatalyst presented higher organoleptic scores, compared to the free cells case, whereas immobilization on BC enhanced probiotic viability during post-fermentation storage (4 °C, 28 days). The obtained high viability (>107 log cfu/g) demonstrated the efficacy of the proposed bioprocess for the production of functional/probiotic-rich beverages. Ultimately, this work presents a consolidated scheme that includes the advantages and the cooperative effect of probiotic LAB strains combined with a functional biopolymer (BC) towards the formulation of novel functional products that coincide with the pillars of food systems sustainability.

Whey protein films reinforced with bacterial cellulose nanowhiskers: Improving edible film properties via a circular economy approach.

Edible films were developed using whey protein concentrate (WPC) and a natural bio-polymer, namely bacterial cellulose (BC). BC was produced via fermentation from orange peels and subsequently acid-hydrolyzed to obtain BC nanowhiskers (BCNW) with high crystallinity (XRD analysis). Morphology of BCNW was analyzed by SEM, TEM, and AFM. WPC/BCNW film composites, containing different amounts of BCNW (0.5-15%, w/w) were developed and characterized. WPC/BCNW film composite was analyzed by Raman spectroscopy, indicating the successful incorporation and the homogenous distribution of BCNW into the WPC film matrix. Mechanical characterization showed that BCNW behaved as a reinforcing filler in the WPC film, increasing tensile strength and Young's modulus by 32% and 80%, respectively. In addition, water vapor permeability was reduced by 33.9% upon the addition of 0.5% BCNW. This study presented a sustainable approach towards the production of WPC films with improved tensile and water barrier properties, suggesting its potential application as a packaging material.

Biosurfactant Production from Lactobacilli: an Insight on the Interpretation of Prevailing Assessment Methods.

Biosurfactants constitute amphiphilic molecules, receiving increased attention as environmentally benign, biodegradable alternatives to substitute for the petroleum derived counterparts in food, pharmaceutical and cosmetics applications. However, their high production cost hinders industrial production. In this study, fifty GRAS lactobacilli strains were screened for their ability to produce biosurfactants, implementing different substrates. Cheese whey permeate (CWP) was also assessed as a low-cost and inherent lactobacilli substrate, aiming to mitigate its polluting impact, expand valorization strategies, alleviate costs deriving from commercial supplements and enhance overall sustainability. Surface tension, emulsification activity (E24) and oil displacement were deployed to identify the most promising candidates. Results reveal surface tension as the most robust method and underline the effect of substrate on biosurfactant synthesis. Likewise, this study indicates the fundamental role of including the final fermentation substrate (CWP) during strain selection to avoid misinterpretation of results and enhance subsequent bioprocess integration.

Psychological burden of covid-19 health crisis on health professionals and interventions to minimize the effect: what has history already taught us?

Health professionals have been at the frontline of the health service since the outbreak of covid-19, responding promptly to diagnose, support and treat infected patients. World Health Organization (WHO) has already praised their contribution and their essential role in controlling this disease. Some of the main concerns of covid-19's impact to health service staff include work overload, exhaustion, and high risk of self-infection or transmission to family members. Moreover, during the pandemic, caregivers' mental health inevitably becomes vulnerable, with salient stress and anxiety-related symptoms. Uncertainty, fear of contagion, guilt, hopelessness, stigmatization and, in some cases, long-term post-traumatic stress disorder (PTSD) are few of the potential effects posed by this outbreak on health workers. In this review, lessons learnt from previous global crises or pandemics on the psychological impact of health workers are presented. History could potentially provide essential information on how to best manage, support and optimize our approach to this highly appreciated and much needed group of professionals. Targeted and prompt interventions could reduce the psychological strain of health professionals, thus, further improving provided patient care. Covid-19 is an on-going health crisis and this work, even though generated by limited existing data, could be used to inform governments and/or institutions and lead on decisions and changes in current guidelines.

Biodiesel production using microbial lipids derived from food waste discarded by catering services.

Biodiesel production using microbial oil derived from food waste discarded by the hospilatity sector could provide a sustainable replacement for diesel fuel. Discarded potato peels were used in solid-state fermentations of Aspergillus awamori for the production of glucoamylase (30 U/g) and protease (50 U/g). Hospitality food waste hydrolysis led to 98% (w/w) starch to glucose conversion yields. Crude hydrolyzates were used in shake flask fermentations with the oleaginous yeast Rhodosporidium toruloides Y-27012 leading to 32.9 g/L total dry weight (TDW) with 36.4% (w/w) intracellular lipid content. Fed-bath bioreactor cultures resulted in TDW of 53.9 g/L and lipid concentration of 26.7 g/L. Principal component analysis showed a fatty acid profile similar to soybean oil and solid food waste oil. Microbial oil was transesterified into biodiesel with satisfactory performance considering the European standard EN 14214. This work demonstrated that valorization of food waste for biodiesel production is feasible.

Olive Oil Oleogel Formulation Using Wax Esters Derived from Soybean Fatty Acid Distillate.

Oleogelation is an emerging technology to structure oils, which can be widely used to substitute saturated and trans fats. Extra virgin olive oil is widely recognized for its high nutritional value, but its utilization in oleogel production is currently limited. In this study, extra virgin olive oil was utilized for the production of a novel oleogel using wax esters derived from soybean fatty acid distillate (SFAD), a byproduct of industrial soybean oil refining. Different concentrations (7%, 10%, 20%, w/w) of SFAD-wax esters were used to evaluate the minimum concentration requirement to achieve oleogelation. Analyses of the mechanical properties of oleogel showed a firmness of 3.8 N, which was then reduced to around 2.1-2.5 N during a storage period of 30 days at 4 °C. Rheological analysis demonstrated that G' is higher than G″ at 20-27 °C, which confirms the solid properties of the oleogel at this temperature range. Results showed that SFAD was successfully utilized for the oleogelation of olive oil, resulting in a novel oleogel with desirable properties for food applications. This study showed that industrial fatty side streams could be reused for the production of value-added oleogels with novel food applications.

Enhanced Aromatic Profile and Functionality of Cheese Whey Beverages by Incorporation of Probiotic Cells Immobilized on Pistacia terebinthus Resin.

: In the present study, cheese whey was utilized for the development of a novel functional beverage, using Lactobacillus casei ATCC 393 probiotic cells immobilized on Pistacia terebinthus resin (pissa Paphos). Evaluation of shelf life of the produced beverages showed that spoilage microorganisms were not observed in beverages containing P. terebinthus resin. Terpenes' rich content might have contributed to the antimicrobial activity of the produced beverages; however, no significant effect on the viability of the immobilized probiotic cells was obtained. Whey beverages containing the immobilized biocatalyst retained a high viability (>1 × 106 CFU/g) of probiotic cells during a storage period of 30 days at 4 °C. The superiority of whey beverages containing the immobilized biocatalyst was also highlighted by GC-MS analysis, while the enhanced aromatic profile, which was mostly attributed to the higher concentration of terpenes, was also detected during the sensory evaluation performed. Conclusively, this study indicated the high commercialization potential of these novel functional whey beverages, within the frame of a sustainable dairy waste valorization approach. To the best of our knowledge, this is the first food-oriented approach within the guidelines of the circular economy reported in the literature, using the autochthonous Pistacia terebinthus resin for the production of functional whey beverages.

Bioprocess development for the production of novel oleogels from soybean and microbial oils.

This study presents the production of novel oleogels via circular valorisation of food industry side streams. Sugarcane molasses and soybean processing side streams (i.e. soybean cake) were employed as fermentation feedstocks for the production of microbial oil. Fed-batch bioreactor fermentations carried out by the oleaginous yeast Rhodosporidium toruloides led to the production of 36.9 g/L total dry weight with an intracellular oil content of 49.8% (w/w) and 89.4 µg/g carotenoids. The carotenoid-rich microbial oil and soybean oil were evaluated as base oils for the production of wax-based oleogels. The wax esters, used as oleogelators, were produced via enzymatic catalysis, using microbial oil or soybean fatty acid distillate as raw materials. All oleogels presented a gel-like behaviour (G' > G″). However, the highest G' was determined for the oleogel produced from soybean oil and microbial oil-wax esters, which indicated a stronger network. Thermal analysis showed that this oleogel had a melting temperature profile up to 35 °C, which is favorable for applications in the confectionery industry. Also, texture analysis demonstrated that soybean oil-microbial oil wax oleogel was stable (1.9-2.2 N) within 30-days storage period. This study showed the potential of novel oleogels production through the development of bioprocesses based on the valorisation of various renewable resources.

Development of a Circular Oriented Bioprocess for Microbial Oil Production Using Diversified Mixed Confectionery Side-Streams.

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6-17 g·L-1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop.

Cheese Whey Processing: Integrated Biorefinery Concepts and Emerging Food Applications.

Cheese whey constitutes one of the most polluting by-products of the food industry, due to its high organic load. Thus, in order to mitigate the environmental concerns, a large number of valorization approaches have been reported; mainly targeting the recovery of whey proteins and whey lactose from cheese whey for further exploitation as renewable resources. Most studies are predominantly focused on the separate implementation, either of whey protein or lactose, to configure processes that will formulate value-added products. Likewise, approaches for cheese whey valorization, so far, do not exploit the full potential of cheese whey, particularly with respect to food applications. Nonetheless, within the concept of integrated biorefinery design and the transition to circular economy, it is imperative to develop consolidated bioprocesses that will foster a holistic exploitation of cheese whey. Therefore, the aim of this article is to elaborate on the recent advances regarding the conversion of whey to high value-added products, focusing on food applications. Moreover, novel integrated biorefining concepts are proposed, to inaugurate the complete exploitation of cheese whey to formulate novel products with diversified end applications. Within the context of circular economy, it is envisaged that high value-added products will be reintroduced in the food supply chain, thereby enhancing sustainability and creating "zero waste" processes.

Evaluation of Biomass and Chitin Production of Morchella Mushrooms Grown on Starch-Based Substrates.

Morchella sp. is one of the most expensive mushrooms with a high nutritional profile. In this study, the polysaccharide content of Morchella species was investigated. Specifically, mycelium growth rate, biomass production, sclerotia formation, and glucosamine and total polysaccharides content of six Morchella species grown on a starch-based media were evaluated. Submerged fermentations in potato dextrose broth resulted in a glucosamine content of around 3.0%. In solid-state fermentations (SSF), using potato dextrose agar, a high linear growth rate (20.6 mm/day) was determined. Increased glucosamine and total polysaccharides content were observed after the formation of sclerotia. Biomass and glucosamine content were correlated, and the equations were used for the indirect estimation of biomass in SSF with agro-industrial starch-based materials. Wheat grains (WG), potato peels (PP), and a mixture of 1:1 of them (WG-PP) were evaluated as substrates. Results showed that the highest growth rate of 9.05 mm/day was determined on WG and the maximum biomass yield (407 mg/g) on WG-PP. The total polysaccharide content reached up to 18.4% of dried biomass in WG-PP. The results of the present study proved encouraging for the efficient bioconversion of potato and other starch-based agro-industrial waste streams to morel biomass and sclerotia eliciting nutritional and bioactive value.

Upgrading Grape Pomace through Pleurotus spp. Cultivation for the Production of Enzymes and Fruiting Bodies.

Grape pomace, a by-product derived from winery industries, was used as fermentation media for the production of added-value products through the cultivation of two Pleurotus species. Solid-state (SSF), semiliquid (SLF), and submerged (SmF) fermentations were carried out using grape pomace as substrate. The effect of the different fermentations on the consumption of phenolic compounds, the production of mycelial mass and enzymes was evaluated using P. ostreatus and P. pulmonarius. The production of fungal biomass and enzymes was influenced by the fermentation mode. The maximum biomass values of ~0.5 g/g were obtained for both P. pulmonarius and P. ostreatus in SmF. Laccase production was induced in SSF and a maximum activity of 26.247 U/g was determined for P. ostreatus, whereas the highest endoglucanase activity (0.93 U/g) was obtained in the SmF of the same fungi. Analysis of phenolic compounds showed that both strains were able to degrade up to 79% of total phenolic content, regardless the culture conditions. Grape pomace was also evaluated as substrate for mushroom production. P. pulmonarius recorded the highest yield and biological efficiency of 14.4% and 31.4%, respectively. This study showed that mushroom cultivation could upgrade winery by-products towards the production of valuable food products.

Probiotics in Food Systems: Significance and Emerging Strategies Towards Improved Viability and Delivery of Enhanced Beneficial Value.

Preserving the efficacy of probiotic bacteria exhibits paramount challenges that need to be addressed during the development of functional food products. Several factors have been claimed to be responsible for reducing the viability of probiotics including matrix acidity, level of oxygen in products, presence of other lactic acid bacteria, and sensitivity to metabolites produced by other competing bacteria. Several approaches are undertaken to improve and sustain microbial cell viability, like strain selection, immobilization technologies, synbiotics development etc. Among them, cell immobilization in various carriers, including composite carrier matrix systems has recently attracted interest targeting to protect probiotics from different types of environmental stress (e.g., pH and heat treatments). Likewise, to successfully deliver the probiotics in the large intestine, cells must survive food processing and storage, and withstand the stress conditions encountered in the upper gastrointestinal tract. Hence, the appropriate selection of probiotics and their effective delivery remains a technological challenge with special focus on sustaining the viability of the probiotic culture in the formulated product. Development of synbiotic combinations exhibits another approach of functional food to stimulate the growth of probiotics. The aim of the current review is to summarize the strategies and the novel techniques adopted to enhance the viability of probiotics.

Biodegradation and toxicity of emerging contaminants: Isolation of an exopolysaccharide-producing Sphingomonas sp. for ionic liquids bioremediation.

Ionic liquids (ILs) have been characterized as contaminants of emerging concern (CEC) that often resist biodegradation and impose toxicity upon environmental release. Sphingomonas sp. MKIV has been isolated as an extreme microorganism capable for biodegradation of major classes of ILs. Six imidazolium-, pyridinium- and ammonium-based ILs (pyridinium trifluoromethanesulfonate [Py][CF3SO3], 1-(4-pyridyl)pyridinium chloride [1-4PPy][Cl], 1-butyl-3-methylimidazolium bromide [BMIM][Br], 1-butyl-3-methylimidazolium methanesulfonate [BMIM][MeSO4], tetrabutylammonium iodide [n-Bu4N][I] and tetrabutylammonium hexafluorophosphate [n-Bu4N][PF6]) were used for microbial growth. The strain achieved 91% and 87% removal efficiency for cultures supplemented with 100 mg L-1 of [BMIM][MeSO4] and [n-Bu4N][I] respectively. The metabolic activity of MKIV was inhibited following preliminary stages of cultures conducted using [BMIM][MeSO4], [BMIM][Br], [Py][CF3SO3] and [n-Bu4N][PF6], indicating potential accumulation of inhibitory metabolites. Thus, a comprehensive toxicological study of the six ILs on Aliivibrio fischeri, Daphnia magna and Raphidocelis subcapitata was conducted demonstrating that the compounds impose moderate and low toxicity. The end-products from [BMIM][MeSO4] and [n-Bu4N][I] biodegradation were assessed using Aliivibrio fischeri, exhibiting increased environmental impact of the latter following biotreatment. MKIV produced 19.29 g L-1 of biopolymer, comprising mainly glucose and galacturonic acid, from 25 g L-1 of glucose indicating high industrial significance for bioremediation and exopolysaccharide production.

Bioprocess development for biolubricant production using microbial oil derived via fermentation from confectionery industry wastes.

Microbial oil produced from confectionery and wheat milling side streams has been evaluated as novel feedstock for biolubricant production. Nutrient-rich fermentation media were produced by a two-step bioprocess involving crude enzyme production by solid state fermentation followed by enzymatic hydrolysis of confectionery industry waste. Among 5 yeast strains and 2 fungal strains cultivated on the crude hydrolysate, Rhodosporidium toruloides and Cryptococcus curvatus were selected for further evaluation for biolubricant production based on fermentation efficiency and fatty acid composition. The extracted microbial oils were enzymatically hydrolysed and the free fatty acids were esterified by Lipomod 34-MDP in a solvent-free system with trimethylolpropane (TMP) and neopentyl glycol (NPG). The highest conversion yields were 88% and 82.7% for NPG esters of R. toruloides and C. curvatus, respectively. This study also demonstrates that NPG esters produced from microbial oil have promising physicochemical properties for bio-based lubricant formulations that could substitute for conventional lubricants.