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.

Integrated biorefinery development using winery waste streams for the production of bacterial cellulose, succinic acid and value-added fractions.

An integrated biorefinery has been developed using winery wastes (grape pomace-GP, stalks-GS, wine lees-WL). Bacterial cellulose was produced from GP extracted free sugars. Grape-seed oil and polyphenols were extracted from GP. Experimental design was employed to optimize lignin removal (50.8%) from mixtures of remaining GP solids and GS via NaOH (1.19% w/v) treatment at 70°C for 30 min. Delignification liquid contained condensed tannins with 76% Stiasny number. Enzymatic hydrolysis produced a sugar-rich hydrolysate (40.2 g/L sugars). Ethanol, antioxidants, tartaric acid and nutrient-rich hydrolysate were produced from WL. The crude hydrolysates were used in fed-batch Actinobacillus succinogenes cultures for 37.2 g/L succinic acid production. The biorefinery produces 42.65 g bacterial cellulose, 24.3 g oil, 40.3 g phenolic-rich extract with 1.41 Antioxidant Activity Index, 80.2 g ethanol, 624.8 g crude tannin extract, 20.03 g tartaric acid and 157.8 g succinic acid from 1 kg of each waste stream.

Immune-Related Gene Expression Profiling of Broiler Chickens Fed Diets Supplemented with Vinification Byproducts: A Valorization Approach II.

The valorization of vinification byproducts portrays a promising bioprocess for the enrichment of animals' diet with bioactive compounds, such as polyphenols, which could regulate the immune response. Therefore, the impact of dietary grounded grape pomace (GGP), wine lees extract (WYC), and grape stem extract (PE) on the relative transcript level of immune related genes of broiler chickens were examined. Two hundred forty, one-day-old as hatched (male/female) chicks (Ross 308) were allocated to four dietary groups, with four replicate pens each with 15 birds. Birds were fed either a basal diet (CON) or the basal diet supplemented with 2.5% GGP, or 0.2% WYC, or 0.1% PE for 42 d. The relative expression of immune-related genes was investigated using a real-time PCR platform. The mRNA levels of Toll-like Receptor 4 (TLR4) were downregulated (p = 0.039) in the liver of broilers fed the GGP-containing diet compared to the CON, while in the spleen of PE-fed broilers, TLR4 was significantly upregulated (p = 0.043). The mRNA levels of interleukin 8 (IL8) tended to upregulate (p = 0.099) in the bursa of Fabricius and were significantly increased (p = 0.036) in the spleen of broilers fed the PE diet. Vinification byproducts depict a promising sustainable source of polyphenols for the poultry feed industry, but more research is needed under field conditions.

Antioxidant Status of Broiler Chickens Fed Diets Supplemented with Vinification By-Products: A Valorization Approach.

Vinification by-products display great potential for utilization as feed additives rich in antioxidant compounds. Thus, the effect of dietary ground grape pomace (GGP), wine lees extract rich in yeast cell walls (WYC), and grape stem extracts (PE) on the relative expression of several genes involved in liver oxidative mechanisms and the oxidative status of the blood and breast muscle of broiler chickens was investigated. In total, 240 one-day-old as hatched chicks (Ross 308) were assigned to four treatments, with four replicate pens and 15 birds in each pen. Birds were fed either a basal diet (CON) or a basal diet supplemented with 25 g/kg GGP, or 2 g/kg WYC, or 1 g starch including 100 mg pure stem extract/kg (PE) for 42 days. The polyphenolic content of vinification by-products was determined using an LC-MS/MS library indicating as prevailing compounds procyanidin B1 and B2, gallic acid, caftaric acid, (+)-catechin, quercetin, and trans-resveratrol. Body weight and feed consumption were not significantly affected. The relative transcript level of GPX1 and SOD1 tended to increase in the liver of WYC-fed broilers, while NOX2 tended to decrease in the PE group. SOD activity in blood plasma was significantly increased in WYC and PE compared to the CON group. The total antioxidant capacity measured with FRAP assay showed significantly higher values in the breast muscle of PE-fed broilers, while the malondialdehyde concentration was significantly decreased in both WYC- and PE-fed broilers compared to the CON group. The exploitation of vinification by-products as feed additives appears to be a promising strategy to improve waste valorization and supply animals with bioactive molecules capable of improving animals' oxidative status and products' oxidative stability.

Volumetric oxygen transfer coefficient as fermentation control parameter to manipulate the production of either acetoin or D-2,3-butanediol using bakery waste.

The formation of either acetoin or D-2,3-butanediol (D-BDO) by Bacillus amyloliquefaciens cultivated on bakery waste hydrolysates has been evaluated in bioreactor cultures by varying the volumetric oxygen transfer coefficient (kLa). The highest D-BDO production (55.2 g L-1) was attained in batch fermentations with kLa value of 64 h-1. Batch fermentations performed at 203 h-1 led to the highest productivity (2.16 g L-1h-1) and acetoin production (47.4 g L-1). The utilization of bakery waste hydrolysate in fed-batch cultures conducted at kLa of 110 h-1 led to combined production of acetoin, meso-BDO and D-BDO (103.9 g L-1). Higher kLa value (200 h-1) resulted to 65.9 g L-1 acetoin with 1.57 g L-1h-1 productivity. It has been demonstrated that the kLa value may divert the bacterial metabolism towards high acetoin or D-BDO production during fermentation carried out in crude bakery waste hydrolysates.

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.

Sustainable production of bio-based chemicals and polymers via integrated biomass refining and bioprocessing in a circular bioeconomy context.

The sustainable production of bio-based chemicals and polymers is highly dependent on the development of viable biorefinery concepts using crude renewable resources for the production of diversified products. Within this concept, this critical review presents the availability of fractionated co-products and fermentable sugars that could be derived from major industrial and food supply chain side streams in EU countries. Fermentable sugars could be used for the production of bio-based chemicals and polymers. The implementation of biorefinery concepts in industry should depend on the evaluation of process efficiency and sustainability including techno-economic, environmental and social impact assessment following circular bioeconomy principles. Relevant sustainability indicators and End-of-Life scenarios have been presented. A case study on the techno-economic evaluation of bio-based succinic acid production from the organic fraction of municipal solid waste has been presented focusing on the evaluation of process profitability and feedstock requirements.

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.

Succinic acid production by immobilized cultures using spent sulphite liquor as fermentation medium.

Spent sulphite liquor (SSL) was used as carbon source for the production of succinic acid using immobilized cultures of Actinobacillus succinogenes and Basfia succiniciproducens on two different supports, delignified cellulosic material (DCM) and alginate beads. Fed-batch immobilized cultures with A. succinogenes in alginates resulted in higher sugar to succinic acid conversion yield (0.81g/g) than the respective yield achieved (0.65g/g) when DCM immobilized cultures were used. The final succinic acid concentration and yield achieved in fed-batch with immobilized cultures of B. succiniciproducens in alginates (45g/L and 0.66g/g) were higher than A. succinogenes immobilized cultures (35.4g/L and 0.61g/g) using nano-filtrated SSL as fermentation medium. Immobilized cultures of B. succiniciproducens in alginate beads were reused in four sequential fed-batch fermentations of nano-filtrated SSL leading to the production of 64.7g of succinic acid with a yield range of 0.42-0.67g/g and productivity range of 0.29-0.65g/L/h. The immobilized cultures improved the efficiency of succinic acid production as compared to free cell cultures.

Evaluation of an integrated biorefinery based on fractionation of spent sulphite liquor for the production of an antioxidant-rich extract, lignosulphonates and succinic acid.

Spent sulphite liquor (SSL) has been used for the production of lignosulphonates (LS), antioxidants and bio-based succinic acid. Solvent extraction of SSL with isopropanol led to the separation of approximately 80% of the total LS content, whereas the fermentations carried out using the pretreated SSL with isopropanol led to the production of around 19g/L of succinic acid by both Actinobacillus succinogenes and Basfia succiniciproducens. Fractionation of SSL via nanofiltration to separate the LS and solvent extraction using ethyl acetate to separate the phenolic compounds produced a detoxified sugar-rich stream that led to the production of 39g/L of succinic acid by B. succiniciproducens. This fractionation scheme resulted also in the production of 32.4g LS and 1.15g phenolic-rich extract per 100g of SSL. Both pretreatment schemes removed significant quantities of metals and heavy metals. This novel biorefinery concept could be integrated in acidic sulphite pulping mills.

Novel technology development through thermal drying of encapsulated Kluyveromyces marxianus in micro- and nano-tubular cellulose in lactose fermentation and its evaluation for food production.

A novel technology development based on the production of a low-cost starter culture for ripening of cheeses and baking is reported in the present study. The starter culture comprises thermally dried cells of Kluyveromyces marxianus encapsulated in micro- and nano-tubular cellulose. For production of a low-cost and effective biocatalyst, whey was used as raw material for biomass production and thermal drying methods (convective, conventional, and vacuum) were applied and evaluated at drying temperatures ranging from 35 to 60 °C. The effect of drying temperature of biocatalysts on fermentability of lactose and whey was evaluated. Storage stability and suitability of biocatalysts as a commercial starter cultures was also assessed and evaluated. All thermally dried biocatalysts were found to be active in lactose and whey fermentation. In all cases, there was sugar conversion ranging from 92 to 100 %, ethanol concentration of up to 1.47 % (v/v), and lactic acid concentrations ranged from 4.1 to 5.5 g/l. However, convective drying of the encapsulated cells of K. marxianus in micro- and nano-tubular cellulose was faster and a more effective drying method while drying at 42 °C appear to be the best drying temperature in terms of cell activity, ethanol, and lactic acid formation. Storage of the biocatalysts for 3 months at 4 °C proved maintenance of its activity even though fermentation times increased by 50-100 % compared with the fresh dried ones.

Scale-up of thermally dried kefir production as starter culture for hard-type cheese making: an economic evaluation.

This paper concerns the effect of thermal-drying methodology on the investment cost for dried kefir cells production in order to be used as starter culture in cheese manufacturing. Kefir cells were produced at pilot plant scale using a 250-L bioreactor and whey as the main substrate. Kefir cells were subsequently dried in a thermal dryer at 38 degrees C and used as a starter culture in industrial-scale production of hard-type cheeses. The use of thermally dried kefir as starter culture accelerated ripening of cheeses by increasing both lipolysis and fermentation rate as indicated by the ethanol, lactic acid, and glycerol formation. Additionally, it reduced coliforms and enterobacteria as ripening proceeded. This constituted the basis of developing an economic study in which industrial-scale production of thermally dried kefir starter culture is discussed. The industrial design involved a three-step process using three bioreactors of 100, 3,000, and 30,000 L for a plant capacity of 300 kg of thermally dried kefir culture per day. The cost of investment was estimated at 238,000 euro, which is the 46% of the corresponding cost using freeze-drying methodology. Production cost was estimated at 4.9 euro/kg of kefir biomass for a 300-kg/day plant capacity, which is the same as with the corresponding cost of freeze-dried cells. However, the estimated added value is up to 10.8 x 10(9) euro within the European Union.

Whey valorisation: a complete and novel technology development for dairy industry starter culture production.

Whey is the major by-product of the dairy industry, produced in large quantities and usually disposed off causing major environmental pollution, due to its high organic load that makes treatment cost prohibitive. This paper comprises a contribution on the valorisation of this high polluting liquid waste of the dairy industry, based on research for the production of novel dairy starter cultures using whey as raw material. Starter cultures are used for cheese ripening in order to: (i) accelerate ripening, (ii) improve quality and (iii) increase shelf-life. The developed technology involves biomass production from whey followed by thermal drying of cultures. Specifically, Kluyveromyces marxianus, Lactobacillus bulgaricus and kefir yeasts were thermally dried, and their efficiency in lactose and milk whey fermentations was studied. The most suitable culture regarding its technological properties was kefir, which was used for cheese ripening in freeze-dried and thermally dried form. Besides the reduction of production cost, which is an essential requirement for the food industry, the use of thermally dried kefir displayed several other advantages such as acceleration of ripening, increase of shelf-life, and improvement of hard-type cheese quality.

Fermentation efficiency of thermally dried kefir.

Three thermal drying methods (conventional, vacuum and convective) were used for drying of kefir biomass and their effect on cell viability, fermentation rate and other kinetic parameters of lactose and whey fermentation were studied. Convective drying rate was higher than conventional and even higher than vacuum at each studied temperature (28, 33 and 38 degrees C). After that, fermentations were performed by kefir biomass dried by the three drying methods. Ethanol concentration, ethanol productivity and ethanol yield are higher in whey fermentations performed by kefir biomass dried with convective drying method. Regarding lactic acid production, fermentation performed by kefir biomass dried with conventional drying method gave higher concentrations, compared to other drying methods. Storage of kefir biomass convectively dried at 33 degrees C for 4months, without any precaution decreases its fermentability and thus reduces ethanol (31%) and lactic acid productivity (20%), but remains a promising technology, since a significant part of its initial fermentative activity is retained.