Increasing Enzyme Dosage Research Articles

A recombinant amylomaltase from Thermus thermophilus STB20 can tailor the macromolecular characteristics of tapioca starch through its transglycosylation activity

Amylomaltase (AM) is a transglucosylase known for producing glucans exclusively with α-1,4 linkages, which is widely used in the industries for modification of starch. In this paper, a study was conducted to investigate both the characteristics of recombinant amylomaltase from Thermus thermophilus STB20 (TtAM) and its impact on the fine structure of tapioca starch. The TtAM showed an excellent thermostability and a high affinity for larger molecular substrates. Changes in the molecular characteristics of tapioca starch were discussed in terms of chain length distribution (CLD) and molecular weight distribution (Mw) under different enzyme dosages and reaction times. The TtAM degraded amylose, which was transferred to short side chains (DP 11-26) of amylopectin, thereby extending short side chains into longer chains (DP >26). The Mw of TtAM-treated starches initially increased and then decreased. With increasing enzyme dosage and reaction time, the TtAM facilitated hydrolysis activities, reducing the proportion of long side chains, which led to increase the dextrose equivalent (DE) value. Cycloamylose (DP 8 - 33) was generated during the modification process, reflecting a diversified range of the enzyme products, and resulting in a polymodal distribution profile in the Mw. These findings potentially broaden the utilization of TtAM in the production of starch conversion products, indicating that novel starch-based derivatives with tailor-made structures can be obtained by controlling the enzyme dosages and reaction times.

LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes

BackgroundHigh substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions.ResultsAlthough glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining).ConclusionsThe gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Filtration enzymes applied during mashing affect beer composition and viscosity

Why was the work done: Filtration enzymes that target the degradation of arabinoxylan and β-glucan are widely used in the brewing industry to improve wort and beer filtration. Although these enzymes have proven their effectiveness in improved lautering efficiency and beer filterability, the effect of varying dosage and type of enzyme preparations on beer composition and quality has not been described.
 How was the work done: The impact of dosage of different filtration enzyme preparations (Laminex®750, Laminex®C2K, and Laminex®MaxFlow4G) was investigated on the free ferulic acid content in the wort, chemical composition and viscosity of beer, together with an emphasis on the content and structure of arabinoxylan and β-glucan.
 What are the main findings: The structural features of arabinoxylan and β-glucan in beer were strongly influenced by the dosage and type of filtration enzyme. In general, the high-molecular weight (HMW) arabinoxylan and HMW β-glucan content, and total β-glucan content in beers decreased with increasing enzyme dosage, while the total arabinoxylan levels increased. The HMW arabinoxylan content was strongly related to beer viscosity. The use of filtration enzymes led to a decreased HMW arabinoxylan content and decreased beer viscosity, which could affect the palate fullness of beers. Overdosing filtration enzymes resulted in more ferulic acid, the precursor (in the presence of phenolic yeast) to the clove-like 4-vinyl guaiacol.
 Why is the work important: This work provides brewers with insight on how filtration enzymes affect beer composition and viscosity. It can help make an informed choice of the type of filtration enzyme and the dosage applied during mashing.

Rheological behavior of high consistency enzymatically fibrillated cellulose suspensions

High-consistency processing of fibrillated cellulose materials is attractive for commercial applications due to potential for lowered production costs, energy savings and easier logistics. The current work investigated structure–property relationships of fibrillated cellulose suspensions produced at 20% consistency using VTT HefCel (High-consistency enzymatic fibrillation of cellulose) technology. Morphological examination of the fibrillated materials revealed that enzymatic action on the cellulose substrates was not a direct function of enzyme dosage but rather was dependent on the raw material composition. Furthermore, shear viscosity of the HefCel suspensions was found to decrease with increasing enzyme dosage while the water retention increased. The shear viscosity followed power law relationship with the power law index varying in the range 0.11–0.73. The shear-thinning behavior decreased with increasing consistency. Moreover, suspension viscosity (upmu) was found to be highly dependent on the consistency (mathrm{c}) as upmu sim {mathrm{c}}^{mathrm{m}}, with mathrm{m} ranging from 2.75 to 4.31 for different samples. Yield stress (τy) of the HefCel suspensions was measured at 7 and 10% consistencies. The performance of the fibrillated cellulose grades in a typical application was demonstrated by casting films, which were characterized for their mechanical properties.Graphic abstract

255 Determining the optimal dose of microbial enzyme to enhance fiber digestion

Abstract Certain enzymes may enhance fiber digestion, but the optimum dosage for ruminants consuming bermudagrass hay is not known. The objective of this study was to determine the optimal dosage of a combination of a protease with Aspergillus oryzae and A. niger fermentation extract blend on fiber digestion and rumen fermentation of bermudagrass hay by sheep. Twenty ewes (30.3 ± 5.29 kg BW) were allocated randomly to 1 of 5 diets (n = 4/treatment). Bermudagrass hay was chopped and offered for ad libitum consumption to ewes housed individually in 1 × 1.5-m pens with plastic-coated grate flooring. Diets were supplemented with 0.25% BW of soybean meal and either no enzyme or 1.5, 3, 4.5, or 6 g of a mixture of a protease with A. oryzae and A. niger fermentation extracts in a calcium carbonate carrier. A 14-d dietary adaptation was followed by 7 d of total fecal collection. On d 21, 3 rumen samples per ewe were taken at 4 h intervals for analysis of pH and VFA concentrations. Data were analyzed using the MIXED procedure of SAS and orthogonal linear and quadratic contrasts were used to assess enzyme dosage effects. Dry matter and ADF digestibility increased quadratically (P < 0.05), and NDF and OM digestibility tended (P ≤ 0.07) to increase quadratically across dosages. Ruminal acetate concentrations decreased quadratically and ruminal propionate increased quadratically (P < 0.05) with increasing enzyme dosage (P < 0.05). Total VFA concentrations were not affected by dosage (P = 0.71), but the acetate:propionate ratio decreased quadratically (P < 0.05) with increasing enzyme dosage. In conclusion, a protease and A. oryzae and A. niger fermentation extract blend increased fiber digestion, but dosages over 3 g of enzyme did not further enhance DMD or DMI.

Comparative life cycle assessment of a waste to ethanol biorefinery system versus conventional waste management methods

Biowaste is a prominent source for second generation biofuels and other biobased products since it constitutes an abundant feedstock, the current treatment of which poses challenges to the environment. In the current paper, the environmental sustainability of a biorefinery system producing ethanol from biowaste is assessed by means of Life Cycle Assessment. It is shown that the biorefinery system presents very good environmental performance, as the net emissions are quite low for all the impact categories examined, while in many of the cases the emissions are negative. The total net global warming impact is estimated to be -15 kg CO2eq/ton biowaste. Following, the environmental impacts from biowaste treatment with the biorefinery system are compared to those from biowaste treatment with the conventional municipal waste management methods. It is shown that the biorefinery system presents very good performance compared to the current waste management methods. Furthermore, a sensitivity analysis is conducted for investigating the impact of increased enzyme dosage to the overall environmental performance of the system. It is found that, the increase in ethanol production due to increased enzyme dosage has a smaller impact to the system performance compared to the effect of increased enzyme dosage. Furthermore, it was shown that the range of enzyme contribution in the global warming impact of ethanol biorefineries is wide (11–62%) which is attributed to a number of parameters, such as the high variation of the reported global warming impact of enzymes, the different enzyme loadings and ethanol yields.

Phosphoric acid-acetone process for cleaner production of acetone, butanol, and ethanol from waste cotton fibers

Abstract To move biobutanol toward a commercialized biofuel, its production process should be developed based on renewable and cost-effective carbon sources. In this study, it was shown that cotton, a commodity that typically ends in landfill sites or incinerators, can be utilized as a feasible source after the phosphoric acid-acetone process. The treatment with phosphoric acid (19 g/g) resulted in 94% cotton dissolution after 60 min, and the addition of acetone (41 g/g) was accompanied by 97% precipitation. Enzymatic hydrolysis of regenerated cellulose (15 filter paper unit per mL (FPU/mL)) followed by fermentation by Clostridium acetobutylicum resulted in 5.4 g/L acetone-butanol-ethanol (ABE). In this process, 208 g acid and 138 FPU cellulase were used for production of each g ABE. Increasing enzyme dosage to 25 FPU/g resulted in 79% higher butanol production (161 g ABE/kg). Through cyclic acid re-concentration, phosphoric acid consumption decreased to 82 g/g ABE. Furthermore, by applying an additional dilute phosphoric acid pretreatment, the enzyme consumption reduced to 113 FPU/g ABE.

Low Molecular Weight Xylanase from Trichoderma viride VKF3 for Bio-bleaching of Newspaper Pulp

Xylanase is a major enzyme used in the paper and pulp industries for bio-bleaching applications. There are possibilities for xylanase with better properties suitable for industrial applications. This paper focused on a potential xylanolytic fungus, Trichoderma viride VKF-3, obtained from a mangrove soil sample. Optimum conditions for xylanase production were tested by culturing T. viride VKF3 under varying carbon and nitrogen sources, medium pH, and incubation temperature. The isolate T. viride VKF3 achieved a maximum of 3.045 IU/mL of xylanase activity by utilizing coconut oil cake as a substrate. During purification, 84% yield was obtained with 40% ammonium sulphate. The enzyme activity was confirmed through zymogram analysis, and a band was observed at 14 kDa. The xylanase facilitated maximum hexenuronic acid release with a 30% enzyme dosage following 4 h of incubation. Moreover, the Kappa number tended to decrease with increased enzyme dosage and incubation time. There was a Δ brightness of 11% following 4 h of enzymatic treatment. Strength properties, such as the tensile, burst indices, and folding endurance, was improved during the xylanase assisted deinking of pulp. Hence, the present xylanase was found to be suitable for the bio-bleaching of newspaper waste via an eco-friendly process.

Optimization and Scale-Up of Enzymatic Hydrolysis of Wood Pulp for Cellulosic Sugar Production

With the decreased demand for pulp and paper worldwide, the reorganization of pulp and paper mills for cellulosic sugar production is possible. To maximize cellulosic sugar production from the wood pulp with minimum resources, the effects of pH, buffer system, temperature, enzyme loadings, pulp concentrations, and mixing modes on enzymatic hydrolysis were investigated, one factor at a time. Temperature played an important role in enzymatic hydrolysis. When the temperature was lower than 45 °C, the sugar production declined dramatically to almost half of the maximum value. Increasing enzyme dosage, increasing pulp concentration, and adding xylanase increased sugar production. The intermittent manual mixing mode generated higher concentrations of sugars and could be used for large-scale production. At pilot-scale, the diverted pulp for the pulping process was directly hydrolyzed without any treatment, and the residue after hydrolysis was hydrolyzed by adding fresh enzymes. This study provides insight on economically feasible enzymatic hydrolysis of wood pulp at large-scale cellulosic sugar production.

Evaluation of Direct Application of Enzymes to Remediate Soil Water Repellency

Organic coatings on sand particles can cause soil water repellency (SWR) where a soil does not spontaneously wet; this leads to challenges in water management and crop production. In laboratory studies, we evaluated a novel approach using direct application of 10 enzymes at three (low, medium, high) dosages to remediate SWR on two sand turfgrass soils in a 3-day incubation study and a second study at high dosage with 1-day incubation. A soil:solution ratio of 1:1 (10 g soil and 10 mL solution) was used and a deionized water control included. For Soil 7, a very strongly hydrophobic soil from a localized dry spot turfgrass area with a water drop penetration time (WDPT) of 7440 seconds (untreated) and 332 to 338 seconds (water-treated), the high dosage rates of laccase, chitinase, and protease at 1 and 3 days incubation resulted in WDPT of less than 60 seconds (i.e., hydrophilic soil). Pectinase exhibited similar results only in the 3-day incubation study. On the strongly hydrophobic Soil 21 (WDPT of 655 seconds untreated; 94 to 133 water-treated) from the dry area of a fairy ring-affected area on a turfgrass site, high dosages of chitinase, laccase, pectinase, and protease reduced WDPT to less than 60 seconds in both studies; and medium dosage rates were also effective for all but protease in the 3-day incubation study. Each of the four most effective enzymes for reducing WDPT, noted previously, demonstrated a significant exponential or logarithmic relationship between decreasing WDPT and increasing enzyme dosage. Further studies in field situations will be required to determine enzyme effectiveness on SWR and water management.

Ethanol fermentation from food processing waste

This study focuses on the use of restaurant waste for production of ethanol. Food wastes (corn, potatoes, and pasta) were converted to ethanol in a two‐step process: a two‐part enzymatic digestion of starch using α‐amylase and glucoamylase and then fermentation of the resulting sugars to ethanol using yeast. Because of the low initial composition of starch in the food waste, low ethanol concentrations were achieved: at best 8 mg/mL ethanol (0.8% by mass). Ethanol concentration increased with increasing enzyme dosage levels. Calculations were conducted to evaluate whether waste heat from restaurant waste could be used to drive flash vaporization to purify ethanol. If the solution produced by fermenting food waste is flashed at a temperature of 99.7°C, 77% of the ethanol is recovered in a vapor stream with 1.14 mol % ethanol (2.87 mass %). Waste heat could provide over a third of the energy for this vaporization process. If 4 mol % ethanol could be produced in the fermentation step by increasing the initial starch content in the waste solution and improving the fermentation process, then a single flash at 98.9°C will recover nearly 99% of the ethanol, giving a mass concentration of ethanol of 10.3%, which is similar to that achieved in industrial grain fermentation. © 2012 American Institute of Chemical Engineers Environ Prog, 32: 1280–1283, 2013

RHEOLOGICAL BEHAVIOR AND PHYSICOCHEMICAL CHARACTERISTICS OF GOLDENBERRY (PHYSALIS PERUVIANA) JUICE AS AFFECTED BY ENZYMATIC TREATMENT

Goldenberry (Physalis peruviana) juice was prepared and enzymatically treated with two dosages (300 and 600 ppm) of Pectinex Ultra SP-L, then, the juice was concentrated to 30 and 40°Brix. Rheological properties of juices were studied at a wide range of temperatures (5 to 100C) and shear rate range from 0.3 to 100/s using Brookfield Digital Viscometer. The Herschel–Bulkley, Casson and Bingham rheological models were applied to describe the rheological properties of different juices. These properties are key parameters required to solve food industry problems in numerous areas such as quality control, evaluation of consumer acceptance and texture. The results indicated that juice concentrates behave as non-Newtonian fluids (pseudoplastic) and have a definite yield stress. The Bingham and Casson, plastic viscosity, yield stress, consistency index and flow index decreased with increasing enzyme dosage and temperature. The effect of temperature on the viscosity described by means of an Arrhenius-type equation. The activation energy for viscous flow depends on the total soluble solids. Moreover, sensory evaluation for juices was carried out. In consideration of industrial utilization, detailed knowledge on the rheological and physicochemical characteristics of cape gooseberry juice is of major importance.

Lipase-Catalyzed Acyl Exchange of Soybean Phosphatidylcholine in n-Hexane: A Critical Evaluation of Both Acyl Incorporation and Product Recovery

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.

The Degree of Redundancy in Metabolic Genes Is Linked to Mode of Metabolism

An understanding of the factors favoring the maintenance of duplicate genes in microbial genomes is essential for developing models of microbial evolution. A genome-scale flux-balance analysis of the metabolic network of Saccharomyces cerevisiae has suggested that gene duplications primarily provide increased enzyme dosage to enhance metabolic flux because the incidence of gene duplications in essential genes is no higher than that in nonessential genes. Here, we used genome-scale metabolic models to analyze the extent of genetic and biochemical redundancy in prokaryotes that are either specialists, with one major mode of energy generation, or generalists, which have multiple metabolic strategies for conservation of energy. Surprisingly, the results suggest that generalists, such as Escherichia coli and Bacillus subtilis, are similar to the eukaryotic generalist, S. cerevisiae, in having a low percentage (<10%) of essential genes and few duplications of these essential genes, whereas metabolic specialists, such as Geobacter sulfurreducens and Methanosarcina barkeri, have a high percentage (>30%) of essential genes and a high degree of genetic redundancy in these genes compared to nonessential genes. Furthermore, the specialist organisms appear to rely more on gene duplications rather than alternative-but-equivalent metabolic pathways to provide resilience to gene loss. Generalists rely more on alternative pathways. Thus, the concept that the role of gene duplications is to boost enzymatic flux rather than provide metabolic resilience may not be universal. Rather, the degree of gene duplication in microorganisms may be linked to mode of metabolism and environmental niche.

Production, purification and characterization of Bacillus sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g−1 bran and 180 IU ml−1, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn2+ and Co2+ increased activity by twofold, while Cu2+ and Fe2+ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K m for oat-spelt xylan was 2.23 mg ml−1 and V max was 296.8 IU mg−1 protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.

Optimization of Reaction Conditions for Enzymatic Viscosity Reduction and Hydrolysis of Wheat Arabinoxylan in an Industrial Ethanol Fermentation Residue

This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 degrees C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, beta-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 degrees C for arabinose release and from pH 4.9-5.3 and 42-46 degrees C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields.

Monitoring of Monooctanoylphosphatidylcholine Synthesis by Enzymatic Acidolysis between Soybean Phosphatidylcholine and Caprylic Acid by Thin-Layer Chromatography with a Flame Ionization Detector

Thin-layer chromatography with a flame ionization detector (TLC-FID) was used for monitoring the production of structured phospholipids (ML type: L, long-chain fatty acids; M, medium-chain fatty acids) by enzyme-catalyzed acidolysis between soybean phosphatidylcholine (PC) and caprylic acid. It was found that the structured PC fractionated into two to three distinct bands on both plate thin-layer chromatography (TLC) and Chromarod TLC. These three bands represented PC of the LL type, ML type, and MM type, respectively. The TLC-FID method was applied in the present study to examine the influence of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio (caprylic acid/PC, mol/mol) on formation of ML-type PC in a batch reactor with Thermomyces lanuginosa lipase as the catalyst. The formation of ML-type PC was dependent on all parameters examined except for the substrate ratio. The ML-type PC content increased with increasing enzyme dosage, reaction temperature, solvent amount, and reaction time. The substrate ratio had no significant effect on the formation of ML-type PC within the tested range (3-15 mol/mol). The formation of MM-type PC was observed in some experiments, indicating that acyl migration is taking place during reaction since the lipase is claimed to be 1,3-specific. The TLC-FID method offers a simple and cheap technique for elucidation of product and byproduct formation during enzyme-catalyzed reactions for production of phospholipids containing mixtures of long- and medium-chain fatty acids.

Effects of different enzymatic pre-press maceration treatments on the release of phenols into blackcurrant juice

The effects of different pectinolytic enzyme treatments on the release of phenolic compounds from blackcurrant berry mash into juice in experimental blackcurrant juice production were examined. The influence of enzyme dose (0–0.1% by weight), degree of berry crushing, maceration time, and temperature on the total phenol concentration, the juice yield, and on polysaccharide degradation were evaluated for four commercial, fungal enzyme preparations in statistically designed experimental templates. In optimal experimental conditions, treatments with Macer8 [FJ] and Pectinex Ultra SP-L released ~6500 and 6650 mg gallic acid equivalents/L of total phenols, respectively. These levels correspond to increases of 14–15% compared to the juice extracted without enzymes, and were significantly higher than those achieved with Rapidase BE Super and Grindamyl pectinase treatments. Increased enzyme dosage gave larger juice yields and higher phenol concentrations. There was a positive, linear correlation between degradation of the substrate polysaccharides and the amount of phenols released. The juice samples inhibited the oxidation of human low-density lipoproteins in vitro in a dose-dependent matter. The non-enzyme-treated sample exhibited higher antioxidant activity than the enzyme-treated juices at equimolar test levels of phenols, presumably because of differences in their phenolic profiles.

Production of inulooligosaccharides from chicory extract by endoinulinase from Xanthomonas oryzae No. 5.

Inulooligosaccharides (IOS) production from chicory extract was carried out using endoinulinase obtained from a new isolate, Xanthomonas oryzae No. 5. The IOS production from chicory extract was maximum when 50 g/liter of chicory extract was utilized as the substrate. As the substrate concentration increased, the IOS production accordingly decreased probably due to substrate inhibition. For a comparative study, enzyme reactions were carried out from pure inulin as substrate. Though total IOS contents indicated higher IOS yield with pure inulin compared to that of chicory extract, the distribution of inulooligosaccharide components between pure inulin and chicory extract was not significantly different; i.e. DP5 and higher oligosaccharides are major products in case of both chicory extract and pure inulin as substrate. A considerable amount of oligofructose (about 30%, w/w), which were originally present in chicory extract, resulted in the change of the enzyme kinetics. A reaction pH 7 was found to be most suitable for enzyme reaction. The initial reaction rates increased with increasing enzyme dosage, although the relative composition of the IOS produced remain unchanged.

Studies on the mode of action of non‐starch‐polysaccharides (NSP)‐degrading enzymes in vitro

By use of an in vitro model, the effects of NSP‐degrading enzymes on the cage effect and the hydration properties were demonstrated using wheat bran. The in vitro model simulates the conditions (pH, dry matter, temperature and transit time) in the fore sections of the porcine gastro‐ intestinal tract (GIT) by neglecting endogenous enzyme activities. Enzyme treatment caused a dose‐dependent increase in wheat bran solubility and thus resulted in improved protein and mineral release from the insoluble NSP fraction. Up to 17% protein and 40% crude ash from the insoluble NSP‐fraction were dissolved after enzyme treatment. Hydrating properties of wheat bran were strongly affected by enzyme treatment and particle size. Water‐binding capacity (WBC) and water‐holding capacity (WHC) decreased with increasing enzyme dosage in dependence on particle size. The studies confirmed the applicability of the tested in vitro model as a useful tool for preliminary tests to estimate the effects of NSP‐degrading enzymes on nutrient release and changes in some physico‐chemical properties.