Optimised production and extraction of astaxanthin from the yeast Xanthophyllomyces dendrorhous

Currently, astaxanthin demand is fulfilled by chemical synthesis using petroleum-based feedstocks. As such, alternative pathways of natural astaxanthin production attracts much research interest. This study aimed at optimising bioreactor operation parameters for astaxanthin production and evaluated strategies for its subsequent extraction. The effect of pH and agitation were evident as significant reduction in both biomass and astaxanthin production was observed when the culture pH was not controlled, and low agitation speed was applied. At controlled pH condition and high agitation speed, significant increase in biomass (16.4 g/l) and astaxanthin production (3.6 mg/l) were obtained. Enzymatic yeast cell lysis using two commercial enzymes (Accellerase 1500 and Glucanex) was optimised using central composite design of experiment (DoE). Accellerase 1500 led to mild cell disruption and only 9% (w/w) of astaxanthin extraction. However, glucanex treatment resulted in complete astaxanthin extractability, compared to standard extraction method (DMSO/acetone). When supercritical CO2 was employed as an extraction solvent in accellerace pre-treated Xanthophyllomyces dendrorhous cells, astaxanthin extraction increased 2.5-fold. Overall, the study showed that extraction conditions can be tailored towards targeted pigments present in complex mixtures, such as in microbial cells

Development of surfactant-coated alginate capsules containing Lactobacillus plantarum

A novel concept is proposed in which alginate capsules containing a model probiotic Lactobacillus plantarum strain are coated with different surfactants with the aim to enhance cell survival during passage initially through simulated gastric (SGF) and then intestinal (SIF) fluid. The surfactants investigated included the anionic sodium dodecyl sulphate (SDS) and ammonium lauryl sulphate (ALS), the cationic dimethyldioctadecylammonium chloride (DDAC), benzalkonium chloride (BZK) and hexadecyltrimethylammonium bromide (CTAB), and the zwitterionic lecithin. Coating the alginate capsules with CTAB, BZK, ALS and SDS resulted in worst survival (~ 4-9 log CFU/g decrease) compared to uncoated capsules (~3 log CFU/g decrease), after 1 hour exposure to SGF and two hours in SIF, which was most likely associated with their gradual penetration inside the microcapsules, as shown by confocal microscopy, and their antimicrobial effects. Coating the alginate capsules with DDAC improved cell survival compared to uncoated capsules (~1.2 CFU/g decrease), whereas coating with lecithin improved cell survival considerably, resulting in almost complete recovery of viable cells in SGF and SIF (~ 0.3 log CFU/g decrease). Although the interaction between alginate and lecithin was relatively weak as demonstrated by turbidity and contact angle measurements, it is likely that the protection was associated with the fact that lecithin was able to penetrate into the capsule rapidly, an observation that was supported by the fact that lecithin enhanced the viability of free cells in SGF and SIF. Lecithin has significant potential of being used as a coating material for probiotic containing capsules

Waste fat biodegradation and biomodification by Yarrowia lipolytica and a bacterial consortium composed of Bacillus spp. and Pseudomonas putida

Fats, oils and greases (FOGs) are a particular environmental threat. Biodegradation of FOGs is a challenge and in this study the biodegradation of waste cooking fats, namely butter and olive oil, was studied using a non-conventional yeast, Yarrowia lipolytica strain LFMB 20, and a bioaugmentation product consisting of Bacillus spp. and Pseudomonas putida CP1 strain. The microorganisms were grown aerobically in shake-flask experiments in an enriched medium supplemented with ca 0.85% w/v of waste fat. Analysis of the remaining substrate showed a removal of ca 90% of the fat by the yeast at the end of the incubation, while the bacteria removed ca 95% of both fats. Growth rate, biomass production and biomass yield per unit of fat consumed were all higher for the yeast compared to the bacterial consortium. The bacterial consortium exhibited autolysis and a significant decrease in its DCW value at the late growth phases of both fat substrate cultures. The main fatty acids (FAs) present in both fats were linoleic (Δ9,12C18:2), oleic (Δ9C18:1), palmitic (C16:0), palmitoleic (Δ9C16:1) and stearic (C18:0) acid. Both the bacterial consortium and Y. lipolytica preferentially removed Δ9C18:1 from the medium, while a negative selectivity against C18:0 was reported. Both inocula produced microbial mass that contained intra-cellular lipid quantities, but the bacterial consortium gave significantly higher lipid in DCW values compared with the yeast (maximum values up to ca 63% w/w for the butter and ca 42% w/w for the olive oil while the respective values for both lipids were 22%±2% w/w for Y. lipolytica). In all cases, intra-cellular lipids in DCW values decreased during the late growth phases, while their FA composition differed with those of the substrate fat

Supercritical fluid extraction of carotenoids from vegetable waste matrices

The aim of this work was to evaluate a previously-developed model on supercritical fluid extraction (SFE) for carotenoid recovery from carrot peels on various carotenoid-rich fruit and vegetable wastes. To this end, 15 matrices, including flesh and peels of sweet potato, tomato, apricot, pumpkin and peach, as well as flesh and wastes of green, yellow and red peppers, were submitted to SFE under optimised conditions (59 °C, 350 bar, 15 g/min CO2, 15.5% (v/v) ethanol as co-solvent, 30 min of extraction time). The obtained extracts were characterised for their total carotenoid content, antioxidant activity and total carotenoid recovery (TCR). TCR values were greater than 90% w/w for most samples, with β-carotene being the most successfully extracted compound (TCRs 88–100% w/w). More polar carotenoids, such as lutein and lycopene, exhibited lower TCRs. A comparison with literature data suggested that carotenoid extraction is partially dependent on the composition of vegetable matrices, specifically on polysaccharide and moisture content. The results indicated that the optimised SFE conditions can be used as a general model for carotenoid extraction from various fruit and vegetable matrices and as a viable method for adding value to these waste streams by generating carotenoid-rich extracts

Evaluation of the prebiotic potential of arabinoxylans extracted from wheat distillers’ dried grains with solubles (DDGS) and in-process samples

Distillers’ dried grains with solubles (DDGS) is a low value agro-industrial by-product, rich in arabinoxylans (AX), which is produced by commercial distillery and bioethanol plants. In a first approach, we investigated the prebiotic potential of four fractions comprising arabinoxylan oligosaccharides (AXOS) and xylo-oligosaccharides (XOS) obtained by enzymatic hydrolysis of AX fractions derived from DDGS and wet solids (in-process sample of DDGS production process)s. Anaerobic batch cultures at in controlled pH conditions were used to test the prebiotic activity of the samples. Results did not show significant differences between the enzymatic treatments used and all AXOS/XOS were extensively fermented after 24 h. In addition, significant increases (P<0.05) in Bifidobacterium and total SCFAs were observed after 24 h of fermentation. Finally, DDGS-derived hydrolysates were separated on an anionic semi-preparative column to prepare AXOS/XOS fractions with degree of polymerisation (DP) greater than 3. Bifidogenic activity and an increase of SCFAs were again observed after 24 h of fermentation, although this time the selectivity was higher and the fermentation slower, suggesting that the fermentation of this substrate could take place (at least partially) in the distal part of the colon with highly desirable beneficial effect

Extractability and characteristics of proteins deriving from wheat DDGS

Wheat Distillers’ Dried Grains with Solubles (DDGS) and in-process samples were used for protein extraction. Prolamins were the predominant protein components in the samples. The absence of extractable α- and γ-gliadins in DDGS indicated protein aggregation during the drum drying processing stage. Prolamin extraction was performed using 70% (v/v) ethanol or alkaline-ethanol solution in the presence of reducing agent. DDGS extracts had relatively low protein contents (14-44.9%, w/w), regardless of the condition applied. The wet solids were the most suitable raw material for protein extraction, with recovery yields of ~ 55% (w/w) and protein content of ~58% (w/w) in 70% (v/v) ethanol. Protein extracts from wet solids were significantly rich in glutamic acid and proline. Mass balance calculations demonstrated the high carbohydrate content (~ 50%, w/w) of solid residues. Overall, the feasibility of utilising in-process samples of DDGS for protein extraction with commercial potential was demonstrated

Valorisation of side streams from wheat milling and confectionery industries for consolidated production and extraction of microbial lipids

Crude enzymes produced via solid state fermentation (SSF) using wheat milling by-products have been employed for both fermentation media production using flour-rich waste (FRW) streams and lysis of Rhodosporidium toruloides yeast cells. Filter sterilization of crude hydrolysates was more beneficial than heat sterilization regarding yeast growth and microbial oil production. The initial carbon to free amino nitrogen ratio of crude hydrolysates was optimized (80.2 g/g) in fed-batch cultures of R. toruloides leading to a total dry weight of 61.2 g/L with microbial oil content of 61.8 % (w/w). Employing a feeding strategy where the glucose concentration was maintained in the range of 12.2 – 17.6 g/L led to the highest productivity (0.32 g/L∙h). The crude enzymes produced by SSF were utilised for yeast cell treatment leading to simultaneous release of around 80% of total lipids in the broth and production of a hydrolysate suitable as yeast extract replacement

Development and characterisation of protein films derived from dried distillers’ grains with solubles and in-process samples

Polymer films were developed utilising proteins extracted from wheat distillers’ dried grains with solubles (DDGS) and in-process samples (wet solids), both by-products of bioethanol production process. Structural characterisation of DDGS and wet solids films indicated a change in the secondary structure of the proteins, reflecting the impact of DDGS production process such as effect of enzyme on protein properties and consequently on the film properties; whereas the developed films exhibited a rough surface with voids. Determination of moisture sensitivity indicated that DDGS films exhibited more hydrophilicity than wet solids films, with the same trend being observed for their water solubility and water uptake. The moisture content and solubility of DDGS films ranged from 10.2-14.2 % and 32.3-41.8 % respectively whereas those for wet solids’ film ranged from 18.9-19.8 % and 23.8-24.2 % respectively. The mechanical properties of DDGS and wet solids (ranging from 0.27-0.32 MPa) were comparatively lower than commercial wheat gluten film (0.6 MPa). The poor mechanical properties and high water vapour permeability of DDGS and the wet solids films limit their application as biodegradable packaging materials. However, based on their hydrophilicity, the developed films have potential applications in agriculture and horticulture as controlled release matrices and soil conditioners

Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production

In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under non-aseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% (w/v). However, 4.0% (w/v) of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% (w/w) per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% (w/w) of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value amongst the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiese

Understanding the influence of processing conditions on the extraction of rhamnogalacturonan-I “hairy” pectin from sugar beet pulp

Sugar beet pectin is rich in rhamnogalacturonan-I (RG-I) region, which is a potential source of prebiotics. RG-I pectin cannot be extracted the same way as commercial homogalacturan-rich pectin using hot acid. Therefore, this study has explored several alternative methods, including microwave-assisted extraction (MAE) and conventional- solvent extraction (CSE) at atmospheric pressure using different solvents, and microwave-assisted hydrothermal extraction (MAHE) under pressure using water. No conclusive differences in microwave and conventional heating were found with heating rate controlled. The optimum treatment times of both MAE and CSE at 90 °C atmospheric pressure and regardless of the solvents used were 120 min; however, MAHE at 130 °C under pressure can dramatically reduce the time to 10 min. Alcohol-insoluble solids (AIS) extracted using pH13 solvent by MAE had both the highest RG-I yield at 25.3% and purity at 260.2 mg/g AIS, followed by AIS extracts using water by MAHE with 7.5% and 166.7 mg/g AIS respectively