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.

Nutritional Improvements of Sourdough Breads Made with Freeze-Dried Functional Adjuncts Based on Probiotic Lactiplantibacillus plantarum subsp. plantarum and Pomegranate Juice.

New types of sourdough breads are proposed, made with freeze-dried sourdough adjuncts based on: (i) Lactiplantibacillus plantarum subsp. plantarum ATCC 14917, a potential probiotic (LP) alone or (ii) with the addition of unfermented pomegranate juice (LPPO) and (iii) pomegranate juice fermented by the same strain (POLP). Physicochemical, microbiological, and nutritional characteristics (in vitro antioxidant capacity, AC, total phenolics, TPC, and phytate content) of the breads were evaluated and compared with commercial sourdough bread. All adjuncts performed well; the best results being those obtained by POLP. Specifically, the highest acidity (9.95 mL of 0.1 M NaOH) and organic acid content (3.02 and 0.95 g/kg, lactic and acetic acid, respectively) as well as better resistance to mold and rope spoilage (12 and 13 days, respectively) were observed for POLP3 bread (sourdough with 6% POLP). Significant nutritional improvements were observed by all adjuncts, in terms of TPC, AC, and phytate reduction (103 mg gallic acid/100 g, 232 mg Trolox/100 g, and 90.2%, respectively, for POLP3). In all cases, the higher the amount of adjunct, the better the results. Finally, the good sensory properties of the products indicate the suitability of the proposed adjuncts for sourdough breadmaking, while their application in freeze-dried, powdered form can facilitate commercial application.

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.

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.

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.

Geographical origin discrimination of "Ntopia" olive oil cultivar from Ionian islands using volatile compounds analysis and computational statistics.

The aim of the present study was to characterize the aroma profile of olive oil of the "Ntopia" (local) cultivar from the Ionian islands (Zakynthos, Kefalonia, Leukada, and Kerkyra) (Greece), and investigate whether specific volatile compounds could be considered as indicators of olive oil geographical origin, using computational statistics. In this context, 137 olive oil samples were subjected to headspace solid phase microextraction coupled to gas chromatography/mass spectrometry using the internal standard method. Computational statistics on the semi-quantitative data of olive oil samples, as rapid machine learning algorithms, showed that specific volatile compounds could be used as indicators of geographical origin of olive oil of the "Ntopia" cultivar, among the four main Ionian islands. Volatile compounds such as ethanol, pentanal, 2,4-dimethylheptane, 3,7-dimethyl-1,3,6-octatriene (E), 2,5-dimethylnonane, 1-hexanol, 6-methyl-5-hepten-2-one, octanal, dl-Limonene, acetic acid hexyl ester and dodecane could aid to the geographical origin discrimination of "Ntopia" olive oil cultivar when two (Zakynthos and Kefalonia) or four (Zakynthos, Kefalonia, Leukada and Kerkyra) Ionian islands are subjected to statistical analysis. The discrimination rate using the cross-validation method was 100% and 85.7%, respectively. These results were further evaluated using training and holdout partitions, during which a comparable classification rate was obtained. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00217-021-03863-2.

Techno-economic evaluation and life-cycle assessment of poly(3-hydroxybutyrate) production within a biorefinery concept using sunflower-based biodiesel industry by-products.

This study presents techno-economic evaluation of a biorefinery concept using biodiesel industry by-products (sunflower meal and crude glycerol) to produce poly(3-hydroxybutyrate) (PHB), crude phenolic extracts (CPE) and protein isolate (PI). The PHB production cost at two annual production capacities ($12.5/kg for 2,500 t PHB/year and $7.8/kg for 25,000 t PHB/year) was not cost-competitive to current PHB production processes when the revenues derived from co-products were not considered. Sensitivity analysis projected the economic viability of a biorefinery concept that could achieve a minimum selling price of $1.1/kg PHB similar to polypropylene. The annual PHB production capacity and the identification of marketable end-uses with respective market prices for the co-products CPE and PI were crucial in attaining process profitability. Greenhouse gas emissions (ca. 0.64 kg CO2-eq/kg PHB) and abiotic depletion potential (61.7 MJ/kg PHB) were lower than polypropylene. Biorefining of sunflower meal and crude glycerol could lead to sustainable PHB production.

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.

Advances in Food and Byproducts Processing towards a Sustainable Bioeconomy.

The bioeconomy concept was initially focused on resource substitution, aiming to mitigate the depletion of fossil resources and confer an alternative approach for resource utilization [...].

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.

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.

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.

Pistacia terebinthus Resin as Yeast Immobilization Support for Alcoholic Fermentation.

A natural resin retrieved from Pistacia terebinthus tree was evaluated as an immobilization carrier of Saccharomyces cerevisiae AXAZ-1 cells targeting successive fermentation batches of sugar synthetic mediums. Fermentation times below 54 h were recorded at temperatures 28-14 °C. In total, 147 compounds were detected using gas chromatography-mass spectrometry (GC-MS) analysis, including alcohols, esters, ketones, aldehydes, acids, and terpenes. Principal component analysis indicated that the state of cells (free/immobilized) and the fermentation temperature primarily affected terpenes' composition. Importantly, no spoilage of the fermented beverages was noted during 90 days of storage at room temperature, most likely due to the high content of extracted terpenoids and phenols (up to 579.01 mg L-1 and 171.8 mg gallic acid equivalent L-1, respectively). Likewise, the developed novel biocatalyst (yeast cells immobilized within Pistacia terebinthus resin) was suitable for the production of low alcohol beverages with an enhanced aromatic profile. The obtained results revealed that the proposed bioprocess shows great commercialization potential in the new fast-growing low-alcohol beverages sector.

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes.

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.