Using pre-fermented sugar beet pulp as a growth medium to produce Pleurotus ostreatus mycelium for meat alternatives.

This study aimed to determine the compatibility of pre-fermented sugar beet pulp to support the growth of Pleurotus ostreatus mycelium in submerged fermentation. The goal was to create a meat alternative based on mycelial-fermented pulp. It was further explored whether pre-fermentation with lactic acid bacteria (LAB) on the pulp increased meat-like properties, such as aroma, springiness, and hardness, in the final product. Three strains were selected from a high throughput screening of 105 plant-derived LAB based on their acidification and metabolite production in the pulp. Two homofermentative strains (Lactococcus lactis) and one heterofermentative strain (Levilactobacillus brevis) were selected based on their low ethanol production, high lactic acid production, and overall acidification of the pulp. Mycelium of P. ostreatus was grown in submerged fermentations on the pre-fermented pulp, and the biomass was removed by centrifugation. The fungal strain consumed all available sugars and acids and released arabinose to the media. Volatiles were detected using GC-MS, and a large increase in concentrations of hexanal, 1-octen-3-ol, and 2-octenal was measured. Concentration of 1-octen-3-ol was lower in the pre-fermented samples vs. the non-pre-fermented. LC-MS amino acid analysis showed the presence of all essential amino acids on day 0 and 7 of fermentation. The highest concentration of amino acids was for glutamic acid/glutamine and aspartic acid/asparagine. A decrease in all amino acids after 7 days of fungal fermentation was measured for all fermentations. The decrease was more significant for pre-fermented samples. This was also confirmed through a total protein determination, except for samples pre-fermented with Lactococcus lactis strain NFICC142 which increased in total protein content after fungal fermentation. The protein digestibility increased after fungal fermentation, and the highest increase was seen for non-pre-fermented samples. The springiness of the fermented product indicated similarities to meat alternatives, while the hardness was much lower than other meat alternatives. The results indicate that dried sugar beet pulp can be used for submerged cultivation of P. ostreatus, but that pre-fermentation does not improve the physical or nutritional properties of the end product significantly, except for an increased protein content for NFICC142 pre-fermented media. This is the first known attempt to use LAB and P. ostreatus in mixed fermentation to produce fungal mycelium, as well as the first attempt at using SBP in a liquid fermentation for mycelial production of P. ostreatus.

Fate of pesticide residues in beer and its by-products.

Sustainable beer production requires a comprehensive assessment of potential hazards such as pesticides in both the finished product and waste streams, as these streams can be used to create high-value by-products. This study presents the tracking of 13 fungicides (azoxystrobin, boscalid, epoxiconazole, fenpropidin, fenpropimorph, fluquiconazole, flutriafol, fluxapyroxad, kresoxym-methyl, spiroxamine, propiconazole, prothioconazole-desthio, and tebuconazole), two insecticides (chlorpyrifos-methyl and deltamethrin), one herbicide (glyphosate), and one growth regulator (mepiquat) through the beer brewing process. Field-treated rye, wheat, and barley samples containing pesticide residues were used as adjunct during brewing. Samples of the beer as well as the by-products (spent grain, spent hops, trub and spent yeast) were collected and extracted with a modified QuEChERS method for pesticide residues analysis using GC-MS/MS and LC-MS/MS. Results show that an average of 58% of pesticide residues are retrieved in the by-products with the highest fraction (53%) recovered in the spent grain, 4% in trub, 1% in spent hops, no residues detected in spent yeast and 9% in the beer. This is consistent with these nonpolar pesticides tending to remain adsorbed to the spent grain during brewing. Glyphosate and mepiquat, the most polar pesticides included in this study, showed a different behavior, with the largest fraction (>80%) being retrieved in sweet wort and transferred to the beer. Processing factors were generated for each pesticide from the adjunct to the beer and to the four by-products.

Treatment with Supercritical CO2 Reduces Off-Flavour of White Alfalfa Protein Concentrate.

White alfalfa protein concentrate from alfalfa (Medicago sativa) is a promising substitute for milk and egg protein due to its functionality. However, it contains many unwanted flavours that limits the amount that can be added to a food without affecting its taste negatively. In this paper, we have demonstrated a simple method for the extraction of white alfalfa protein concentrate followed by a treatment with supercritical CO2. Two concentrates were produced at lab scale and pilot scale, with yields of 0.012 g (lab scale) and 0.08 g (pilot scale), of protein per g of total protein introduced into the process. The solubility of the protein produced at lab scale and pilot scale was approximately 30% and 15%, respectively. By treating the protein concentrate at 220 bar and 45 °C for 75 min with supercritical CO2, off-flavours were lowered. The treatment did not decrease the digestibility or alter the functionality of white alfalfa protein concentrate when it was used to substitute egg in chocolate muffins and egg white in meringues.

Valorization of Green Biomass: Alfalfa Pulp as a Substrate for Oyster Mushroom Cultivation.

In this study, the potential of alfalfa pulp as an alternative substrate to wheat straw for the cultivation of oyster mushroom (Pleurotus ostreatus) was investigated. The major components associated with different mushroom stages were evaluated, as well as changes in lignocellulolytic enzyme activities in substrates composed of alfalfa pulp, wheat straw or a combination of both. Based on the results, alfalfa pulp was demonstrated to be a better substrate than wheat straw for the production of oyster mushrooms, with a high biological efficiency of 166.3 ± 25.4%. Compared to the cultivation period on commercial straw (31 days), a shorter lifecycle for oyster mushroom was found on alfalfa pulp (24 days), which could help to reduce the risk of contamination during industrial production. Study of the spent substrate as well as the harvested mushrooms revealed that the biological efficiency was related to the higher protein content (17.42%) in the alfalfa pulp compared to wheat straw, as well as greater degradation of cellulose (57.58%) and hemicellulose (56.60%). This was, by and large, due to greater extracellular hydrolytic and oxidative enzyme activity from the mushroom growth in the alfalfa pulp. The quality and safety of the fruiting bodies produced on alfalfa pulp was evaluated, which showed that the protein content was 20.4%, of which 46.3% was essential amino acids, and levels of trace elements and heavy metals were below acceptable limits. Hence, oyster mushroom cultivation using alfalfa pulp provides an alternative method to produce a value-added product, while reducing the biomass wastes in the green protein bio-refinery, and may contribute to sustainable growth in the agricultural industry.

Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis.

There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity.

Scale-Up of Alfalfa (Medicago sativa) Protein Recovery Using Screw Presses.

As a consequence of the increased demand for proteins for both feed and food, alternative protein sources from green plants such as alfalfa (Medicago sativa) have come into focus, together with methods to recover these proteins. In this study, we have investigated the use of screw presses for protein recovery from alfalfa at laboratory and pilot scale. We found that using a pilot scale screw press, with a working pressure of 6 bar, 16% of the total protein was recovered in one pressing, and that after rehydrating and repressing the alfalfa up to ten times, 48% of the total protein could be recovered. The green alfalfa protein concentrate was analyzed for total protein, amino acid profile, protein digestibility, color, ash, fiber and fat content. It was found that repetitive pressings lowered the digestibility of the protein pool and reduced the total protein concentration due to dilution. To achieve the best quality protein at the highest concentrations, it is recommended to press the alfalfa no more than twice, which results in an alfalfa protein concentrate with more than 32% soluble protein and greater than 82% digestibility.

Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures.

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man5GlcNAc2 glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man3-5GlcNAc2 species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man3GlcNAc2 glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex6-7HexNAc2 structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man3-5GlcNAc2 species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles.

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co2+ and inhibited by Mg2+, K+ and Ca2+ followed by Zn2+, Na+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation.

Effects of dissolved oxygen concentration and iron addition on immediate-early gene expression of Magnetospirillum gryphiswaldense MSR-1.

We report the effects of dissolved oxygen (DO) concentration and iron addition on gene expression of Magnetospirillum gryphiswaldense MSR-1 cells during fermentations, focusing on 0.25-24 h after iron addition. The DO was strictly controlled at 0.5% or 5% O2, and compared with aerobic condition. Uptake of iron (and formation of magnetosomes) was only observed in the 0.5% O2 condition where there was little difference in cell growth and carbon consumption compared to the 5% O2 condition. Quantitative reverse transcription PCR analysis showed a rapid (within 0.25 h) genetic response of MSR-1 cells after iron addition for all the genes studied, except for MgFnr (oxygen sensor gene) and fur (ferric uptake regulator family gene), and which in some cases was oxygen dependent. In particular, expression of sodB1 (superoxide dismutase gene) and feoB1 (ferrous transport protein B1 gene) was markedly reduced in cultures at 0.5% O2 compared to those at higher oxygen tensions. Moreover, expression of katG (catalase-peroxidase gene) and feoB2 (ferrous transport protein B2 gene) was reduced markedly by iron addition, regardless of oxygen conditions. These data provide a greater understanding of molecular response of MSR-1 cells to environmental conditions associated with oxygen and iron metabolisms, especially relevant to immediate-early stage of fermentation.

Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml(-1)) were obtained with media containing soy peptone (3-6 g l(-1)) and glutamate (3.6 g l(-1)). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml(-1)) were also much greater (8-150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.

Covalent immobilization of ß-glucosidase on magnetic particles for lignocellulose hydrolysis.

ß-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, ß-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-ß-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified ß-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized ß-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized ß-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized ß-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

Examining the potential of plasma-assisted pretreated wheat straw for enzyme production by Trichoderma reesei.

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml(-1)) and cellulases (0.5 filter paper units (FPU) ml(-1)). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml(-1)) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml(-1)) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.

Integrated processing and multiple re-use of immobilised lipase by magnetic separation technology.

High-gradient magnetic separation based processing is demonstrated for the semi-continuous multicycle re-use of a lipase immobilised on magnetic microparticles. The lipase of Candida antarctica A-type (CALA) was immobilised on polyvinyl alcohol coated magnetic particles (1-2mum diameter) with epoxy functionalisation. The immobilised CALA was used to hydrolyse a model oil-water 2-phase-system composed of a phosphate buffer with tributyrin at up to 3l scale. The immobilised enzyme was subsequently recovered in a magnetic filter using high-gradient magnetic separation and reapplied in repeated cycles of hydrolysis and recovery. Two different temperatures of 30 and 50 degrees C, tributyrin concentrations (0.12 and 35gl(-1)) and reaction times were tested. In each case the reaction was followed by pH titration using NaOH, as well as by HPLC analysis. Consecutive cycles were conducted for each reaction condition and in total the immobilised CALA was subjected to 20 recovery and re-use cycles, after which approximately 14% of the initial specific activity still remained.

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles.

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01-0.2 mg ml(-1)). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.