Recovery of Bioactive Compounds from Industrial Exhausted Olive Pomace through Ultrasound-Assisted Extraction.

Exhausted olive pomace (EOP) is the main agro-industrial waste of the olive pomace extracting industries. It contains phenolic compounds and mannitol, so the extraction of these bioactive compounds should be considered as a first valorization step, especially if EOP is used as biofuel. Therefore, EOP was subjected to bath-type ultrasound-assisted extraction (UAE), and the effects of the acetone concentration (20-80%, v/v), solid load (2-15%, w/v), and extraction time (10-60 min) on the extraction of antioxidant compounds were evaluated according to a Box-Behnken experimental design. By means of the response surface methodology, the optimum conditions were obtained: 40% acetone, 8.6% solids, and 43 min. For all the extracts, the total phenolic content (TPC), flavonoid content (TFC), and antioxidant activity (DPPH, ABTS, and FRAP) were determined. With the aim of shortening the extraction time, a two-level factorial experiment design was also carried out using a probe-type UAE, keeping the solid load at 8.6% (w/v) and the acetone concentration at 40% (v/v), while the amplitude (30-70%) and the extraction time (2-12 min) were varied to maximize the aforementioned parameters. Finally, a maximum of phenolic compounds was reached (45.41 mg GAE/g EOP) at 12 min and 70% amplitude. It was comparable to that value obtained in the ultrasonic bath (42.05 mg GAE/g EOP), but, remarkably, the extraction time was shortened, which translates into lower costs at industrial scale. Moreover, the bioactive compound hydroxytyrosol was found to be the major phenolic compound in the extract, i.e., 5.16 mg/g EOP (bath-type UAE) and 4.96 mg/g EOP (probe-type UAE). Other minor phenolic compounds could be detected by capillary zone electrophoresis and liquid-chromatography-mass spectrometry. The sugar alcohol mannitol, another bioactive compound, was also found in the extract, and its content was determined. Thus, the use of this technology can support the valorization of this waste to obtain bioactive compounds, including mannitol, hydroxytyrosol, and other derivatives, before being applied for other uses.

The use of flow cytometry to assess Rhodosporidium toruloides NCYC 921 performance for lipid production using Miscanthus sp. hydrolysates.

The yeast Rhodosporidium toruloides NCYC 921 was used for lipid production, using Miscanthus biomass hydrolysate as carbon source. The hydrolysate was obtained by enzymatic hydrolysis of Miscanthus biomass (at high solids loading) previously subjected to a hydrothermal pre-treatment. Afterwards R. toruloides was grown on Miscanthus sp. hydrolysate (MH), undiluted and diluted, at the ratios of 1:4 (20 % v/v), 1:2 (33.3 % v/v) and 3:1 (75 % v/v). The best yeast performance was observed for MH 1:2 medium dilution, reaching the maximal biomass concentration of 6.3 g/L, the lipid content of 30.67 % w/w dry cell weight and the lipid concentration of 1.64 g/L. Flow cytometry demonstrated that R. toruloides cell membrane was massively damaged when the yeast was grown on undiluted MH, due to the presence of phenolic compounds; however, when the yeast was grown on diluted MH 1:2 and 1:4, the proportion of intact cells has increased during the yeast cultivation.

Assessment of the effect of autohydrolysis treatment in banana's pseudostem pulp.

Banana's pseudostem pulp (BPP) is a potential by-product obtained in the mechanical fiber extraction of banana's pseudostem. Its chemical characterization revealed to have an interesting composition, with a high polysaccharides content and low content in lignin, which makes it particularly relevant for the biorefinery's biochemical platform. Autohydrolysis pretreatment, studied under isothermal (140 °C) and non-isothermal conditions (140-220 °C), yielded oligosaccharides, mainly gluco-oligosaccharides, as the main soluble products. The highest oligosaccharides production (24 g/100 g raw material) was obtained at a severity factor of 2.3. Autohydrolysis pretreatment effectively disrupted the structure of the material, inducing an improvement of the enzymatic digestibility from 73% for the raw material up to 90% for the most severe conditions. Two stage autohydrolysis, with increasing severity, was also studied, allowing to obtain a higher amount of oligosaccharides (32 g/100 g raw material) and higher digestibility of the remaining solid (up to 97%).

Assessment of the bifidogenic effect of substituted xylo-oligosaccharides obtained from corn straw.

This work evaluates the bifidogenic potential of substituted xylo-oligosaccharides (XOS) obtained from a lignocellulosic feedstock (corn straw). Autohydrolysis was used to selectively hydrolyse the xylan-rich hemicellulosic fraction and the soluble oligosaccharides were purified by gel filtration chromatography. Selected oligosaccharides fractions within the target ranges of polymerization degree (4-6 and 9-21, samples S1 and S2, respectively) were characterized and their bifidogenic potential was investigated by in vitro fermentations using human fecal inocula. Bacterial growth was assessed by fluorescent in situ hybridization (FISH). XOS consumption and short-chain fatty acids (SCFA) production were evaluated and compared with commercial oligosaccharides. Under the tested conditions, all the substrates were utilized by the microbiota, and fermentation resulted in increased bifidobacteria populations. Samples S1 and S2 increased bifidobacteria populations and the production profile of SCFA was similar for XOS samples and commercial oligosaccharides although XOS samples displayed the highest concentration of SCFA on longer fermentation times.

Pulp properties resulting from different pretreatments of wheat straw and their influence on enzymatic hydrolysis rate.

Wheat straw was subjected to three different processes prior to saccharification, namely alkaline pulping, natural pulping and autohydrolysis, in order to study their effect on the rate of enzymatic hydrolysis. Parameters like medium concentration, temperature and time have been varied in order to optimize each method. Milling the raw material to a length of 4mm beforehand showed the best cost-value-ratio compared to other grinding methods studied. Before saccharification the pulp can be stored in dried form, leading to a high yield of glucose. Furthermore the relation of pulp properties (i.e. intrinsic viscosity, Klason-lignin and hemicelluloses content, crystallinity, morphology) to cellulose hydrolysis is discussed.

Hydrolysis of oligosaccharides over solid acid catalysts: a review.

Mild fractionation/pretreatment processes are becoming the most preferred choices for biomass processing within the biorefinery framework. To further explore their advantages, new developments are needed, especially to increase the extent of the hydrolysis of poly- and oligosaccharides. A possible way forward is the use of solid acid catalysts that may overcome many current drawbacks of other common methods. In this Review, the advantages and limitations of the use of heterogeneous catalysis for the main groups of solid acid catalysts (zeolites, resins, carbon materials, clays, silicas, and other oxides) and their relation to the hydrolysis of model soluble disaccharides and soluble poly- and oligosaccharides are presented and discussed. Special attention is given to the hydrolysis of hemicelluloses and hemicellulose-derived saccharides into monosaccharides, the impact on process performance of potential catalyst poisons originating from biomass and biomass hydrolysates (e.g., proteins, mineral ions, etc.). The data clearly point out the need for studying hemicelluloses in natura rather than in model compound solutions that do not retain the relevant factors influencing process performance. Furthermore, the desirable traits that solid acid catalysts must possess for the efficient hemicellulose hydrolysis are also presented and discussed with regard to the design of new catalysts.

Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre by a non-isothermal process.

Oil palm empty fruit bunches (OPEFB) fibre, a by-product generated from non-woody, tropical perennial oil palm crop was evaluated for xylooligosaccharides (XOS) production. Samples of OPEFB fibre were subjected to non-isothermal autohydrolysis treatment using a temperature range from 150 to 220 °C. The highest XOS concentration, 17.6g/L which relayed from solubilisation of 63 g/100 g xylan was achieved at 210 °C and there was a minimum amount of xylose and furfural being produced. The chromatographic purification which was undertaken to purify the oligosaccharide-rich liquor resulted in a product with 74-78% purity, of which 83-85% was XOS with degree of polymerisation (DP) between 5 and 40.

Pre-treatment of lignocellulosic biomass using ionic liquids: wheat straw fractionation.

This work is devoted to study pre-treatment methodologies of wheat straw with 1-ethyl-3-methylimidazolium acetate ([emim][CH3COO]) and subsequent fractionation to cellulose, hemicellulose and lignin. The method developed and described here allows the separation into high purity carbohydrate and lignin fractions and permits an efficient IL recovery. A versatility of the established method was confirmed by the IL reuse. The fractionation of completely dissolved biomass led to cellulose-rich and hemicellulose-rich fractions. A high purity lignin was also achieved. To verify the potential further applicability of the obtained carbohydrate-rich fractions, and to evaluate the pre-treatment efficiency, the cellulose fraction resulting from the treatment with [emim][CH3COO] was subjected to enzymatic hydrolysis. Results showed a very high digestibility of the cellulose samples and confirmed a high glucose yield for the optimized pre-treatment methodology.

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains.

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⁻¹ for L. acidophilus, with a cell mass yield (Y (X/S)) of 0.37 g g⁻¹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⁻¹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis-Menten constant (K(m)) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⁻¹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K(m) for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.

In vitro evaluation of the fermentation properties and potential prebiotic activity of caprine cheese whey oligosaccharides in batch culture systems.

The prebiotic effect of oligosaccharides recovered and purified from caprine whey, was evaluated by in vitro fermentation under anaerobic conditions using batch cultures at 37°C with human faeces. Effects on key gut bacterial groups were monitored over 24 h by fluorescence in situ hybridization (FISH), which was used to determine a quantitative prebiotic index score. Production of short-chain fatty acids (SCFAs) as fermentation end products was analyzed by high-performance liquid chromatography (HPLC). Growth of Bifidobacterium spp was significantly higher (P ≥ 0.05) with the purified oligosaccharides compared to the negative control. Lactic and propionic acids were the main SCFAs produced. Antimicrobial activity of the oligosaccharides was also tested, revealing no inhibition though a decrease in Staphylococcus aureus and Escherichia coli growth. These findings indicate that naturally extracted oligosaccharides from caprine whey could be used as new and valuable source of prebiotics.

Mannitol production by lactic acid bacteria grown in supplemented carob syrup.

Detailed kinetic and physiological characterisation of eight mannitol-producing lactic acid bacteria, Leuconostoc citreum ATCC 49370, L. mesenteroides subsp. cremoris ATCC19254, L. mesenteroides subsp. dextranicum ATCC 19255, L. ficulneum NRRL B-23447, L. fructosum NRRL B-2041, L. lactis ATCC 19256, Lactobacillus intermedius NRRL 3692 and Lb. reuteri DSM 20016, was performed using a carob-based culture medium, to evaluate their different metabolic capabilities. Cultures were thoroughly followed for 30 h to evaluate consumption of sugars, as well as production of biomass and metabolites. All strains produced mannitol at high yields (>0.70 g mannitol/g fructose) and volumetric productivities (>1.31 g/l h), and consumed fructose and glucose simultaneously, but fructose assimilation rate was always higher. The results obtained enable the studied strains to be divided mainly into two groups: one for which glucose assimilation rates were below 0.78 g/l h (strains ATCC 49370, ATCC 19256 and ATCC 19254) and the other for which they ranged between 1.41 and 1.89 g/l h (strains NRRL B-3692, NRRL B-2041, NRRL B-23447 and DSM 20016). These groups also exhibited different mannitol production rates and yields, being higher for the strains with faster glucose assimilation. Besides mannitol, all strains also produced lactic acid and acetic acid. The best performance was obtained for L. fructosum NRRL B-2041, with maximum volumetric productivity of 2.36 g/l h and the highest yield, stoichiometric conversion of fructose to mannitol.

Dilute acid hydrolysis of wheat straw oligosaccharides.

The dilute acid posthydrolysis of wheat straw hemicellulosic oligosaccharides obtained by autohydrolysis was evaluated. An empirical model was used to describe the effect of catalyst concentration (sulfuric acid, 0.1-4% w/w) and reaction time (0-60 min) based on data from a Doehlert experimental design. Catalyst concentration is the main variable influencing posthydrolysis performance, as both its linear and quadratic coefficients are statistically significant for the majority of the studied variables, namely, the ones related to sugar and byproducts production. Reaction time influences xylose and furan derivatives concentrations but not phenolics or acetic acid content. Catalyst concentration and reaction time interact synergistically, minimizing sugar recovery and promoting furan derivatives production. Based on the proposed models, it was possible to delimit an operational range that enables to obtain high monosaccharides recovery together with a slight decrease in inhibitors content as compared to the standard acid hydrolysis treatment. Furthermore, this is achieved with up to 70% less acid spending or considerable savings on reaction time.

Wheat straw autohydrolysis: process optimization and products characterization.

Wheat straw was subjected to autohydrolysis treatments in order to selectively hydrolyze the hemicellulose fraction. The effects of temperature (150-240 degrees C) and non-isothermal reaction time on the composition of both liquid and solid phases were evaluated and interpreted using the severity factor (log R0). The operational conditions leading to the maximum recovery of hemicellulose-derived sugars were established for log R0 = 3.96 and correspond to 64% of the original (arabino)xylan with 80% of sugars as xylooligosaccharides. Under these conditions, a solubilization of 58% xylan, 83% arabinan, and 98% acetyl groups occurred. Glucan was mainly retained in the solid phase (maximum solubilization 16%), which enables an enrichment of the solid phase to contain up to 61% glucan. Delignification was not extensive, being utmost 15%. The yields of soluble products, including sugars, acetic acid, and degradation compounds, such as, furfural, 5-hydroxymethylfurfural furfural obtained suggest the fitness of liquid stream for fermentation purposes or to obtain xylooligosaccharides with potential applications in food, pharmaceutical, and cosmetic industries.

Yeast biomass production in brewery's spent grains hemicellulosic hydrolyzate.

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h(-1), 0.61 g g(-1), and 0.56 g l(-1) h(-1), respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

Biotechnological valorization potential indicator for lignocellulosic materials.

This report introduces the biotechnological valorization potential indicator (BVPI) concept, a metric to measure the degree of suitability of lignocellulosic materials to be used as feedstock in a biorefinery framework. This indicator groups the impact of the main factors influencing upgrade-ability, both the biological/chemical nature of the materials, and the economical, technological and geographical factors. The BVPI was applied to the identification of the most relevant opportunities and constraints pertaining to the lignocellulosic by-products from the Portuguese agro-industrial cluster. Several by-products were identified with a high valorization potential, e.g., rice husks, brewery's spent grain, tomato pomace, carob pulp, de-alcoholized grape bagasse, and extracted olive bagasse, that would greatly benefit from the further development of specific biotechnology processes, specifically concerning the upgrade of their hemicellulosic fraction.

Xylitol production by Debaryomyces hansenii in brewery spent grain dilute-acid hydrolysate: effect of supplementation.

A brewery spent-grain hemicellulosic hydrolysate was used for xylitol production by Debaryomyces hansenii. Addition of 6 g yeast extract/l increased the xylitol yield to 0.57 g/g, and productivity to 0.51 g/l h that were, respectively, 1.4 -and 1.8-times higher than the values obtained with non-supplemented hydrolysate. When corn steep liquor was combined with 3 g yeast extract/l, the highest xylitol yield, 0.58 g/g, was obtained with a similar productivity.

The combined effects of acetic acid, formic acid, and hydroquinone on Debaryomyces hansenii physiology.

The combined effects of inhibitors present in lignocellulosic hydrolysates was studied using a multivariate statistical approach. Acetic acid (0-6 g/L), formic acid (0-4.6 g/L), and hydroquinone (0-3 g/L) were tested as model inhibitors in synthetic media containing a mixture of glucose, xylose, and arabinose simulating concentrated hemicellulosic hydrolysates. Inhibitors were consumed sequentially (acetic acid, formic acid, and hydroquinone), alongside to the monosaccharides (glucose, xylose, and arabinose). Xylitol was always the main metabolic product. Additionally, glycerol, ethanol, and arabitol were also obtained. The inhibitory action of acetic acid on growth, on glucose consumption and on all product formation rates was found to be significant (p < or = 0.05), as well as formic acid inhibition on xylose consumption and biomass production. Hydroquinone negatively affected biomass productivity and yield, but it significantly increased xylose consumption and xylitol productivity. Hydroquinone interactions, either with acetic or formic acid or with both, are also statistically significant. Hydroquinone seems to partially lessen the acetic acid and amplify formic acid effects. The results clearly indicate that the interaction effects play an important role on the xylitol bioprocess.

Effects of aliphatic acids, furfural, and phenolic compounds on Debaryomyces hansenii CCMI 941.

Debaryomyces hansenii is a polyol overproducing yeast that can have a potential use for upgrading lignocellulosic hydrolysates. Therefore, the establishment of its tolerance to metabolic inhibitors found in hydrolysates is of major interest. We studied the effects of selected aliphatic acids, phenolic compounds, and furfural. Acetic acid favored biomass production for concentrations <6.0 g/L. Formic acid was more toxic than acetic acid and induced xylitol accumulation (maximum yield of 0.21 g/g of xylose). All tested phenolics strongly decreased the specific growth rate. Increased toxicity was found for hydroquinone, syringaldehyde, and 4-methylcatechol and was correlated to the compound's hydrophobicity. Increasing the amount of furfural led to longer lag phases and had a detrimental effect on specific growth rate and biomass productivity.

Comparison of two posthydrolysis processes of Brewery's spent grain autohydrolysis liquor to produce a pentose-containing culture medium.

A readily fermentable pentose-containing hydrolysate was obtained from Brewery's spent grain by a two-step process consisting of an auto-hydrolysis (converting the hemicelluloses into oligosaccharides) followed by an enzymatic or sulfuric acid-catalyzed posthydrolysis (converting the oligosaccharides into monosaccharides). Enzymatic hydrolyses were performed with several commercial enzymes with xylanolytic and cellulolytic activities. Acid-catalyzed hydrolyses were carried out at 121 degrees C under various sulfuric acid concentrations and reaction times, and the effects of treatments were interpreted by means of a corrected combined severity factor (CS*), which varied in the range of 0.80-2.01. Under the tested conditions, chemical hydrolysis allowed higher pentose yields than enzymatic hydrolysis. Optimized conditions (defined by CS* = 1.10) allowed both complete monosaccharide recovery and low content of inhibitors. Liquors subjected to posthydrolysis under optimal conditions were easily fermented by Debaryomyces hansenii CCMI 941 in semiaerobic shake-flask experiments, leading to xylitol and arabitol as major fermentation products. The bioconversion process was improved by hydrolysate concentration and supplementation of fermentation media with casamino acids.

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media.

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39-3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10:67:32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars, furfural, HMF, and acetic acid with high biomass yield, 0.68 C-mol/C-mol, and productivity, 0.92 g/(L.h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).