Production of Modified Nucleosides in a Continuous Enzyme Membrane Reactor.

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review.

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

Enzymatic Synthesis of Human Milk Fat Substitute - A Review on Technological Approaches.

Human milk fat substitute (HMFS) is a structured lipid designed to resemble human milk fat. It contains 60-70% palmitic acid at the sn-2 position and unsaturated fatty acids at the sn-1,3 positions in triacylglycerol structures. HMFS is synthesized by the enzymatic interesterification of vegetable oils, animal fats or a blend of oils. The efficiency of HMFS synthesis can be enhanced through the selection of appropriate substrates, enzymes and reaction methods. This review focuses on the synthesis of HMFS by lipase-catalyzed interesterification and provides a detailed overview of biocatalysts, substrates, synthesis methods, factors influencing the synthesis and purification process for HMFS production. Major challenges and future research in the synthesis of HMFS are also discussed. This review can be used as an information for developing future strategies in producing HMFS.

Increased γ-Aminobutyric Acid Content of Germinated Brown Rice Produced in Membrane Reactor.

RESEARCH BACKGROUND: Rice germination is a natural approach to enhance the physical and functional properties of brown rice. Thus, the aim of this study is to investigate the influence of different germination methods on functional properties of germinated brown rice and evaluate the process feasibility. EXPERIMENTAL APPROACH: Brown rice of IPB 3S variety was germinated with three different methods: (i) complete soaking without water replacement, (ii) complete soaking with water replacement every six hours, and (iii) complete soaking with continuous washing in the developed membrane-facilitated soaking reactor. RESULTS AND CONCLUSIONS: The application of the membrane reactor for producing germinated brown rice maintained the pH of the soaking solution relatively constant (i.e. 6.8-7.0). This indicated the circumvention of natural fermentation during brown rice germination. Moreover, the mass fraction of γ-aminobutyric acid in germinated brown rice produced in the membrane reactor was about 4.5-fold higher (169.2 mg/100 mg) than in ungerminated brown rice (36.82 mg/100 mg), and also higher than that of the other two soaking methods. The γ-oryzanol mass fractions and the antioxidant capacity expressed as ascorbic acid equivalents of germinated brown rice obtained with the three soaking methods varied from 32 to 38 mg/100 mg and 18 to 28 mg/100 g, respectively. Within this study, germination could also slightly reduce the transition temperatures of germinated brown rice starch gelatinization (t o=73-74 °C, t p=76-77 °C and t c=~80 °C, where t o, t p and t c are onset, peak and conclusion (final) temperatures). In conclusion, the production of germinated brown rice in the membrane reactor could enhance its γ-aminobutyric acid mass fraction and reduce wastewater production and is therefore considered more feasible. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study demonstrates the feasibility of germinated brown rice production using a membrane-facilitated soaking reactor with enhancement of bioactive compound content, especially γ-aminobutyric acid, and minimised wastewater production.

The utilization of bilimbi (Averrhoa bilimbi) and lime (Citrus aurantifolia) juices as natural acid coagulants for tofu production.

The influences of coagulation conditions on the characteristics of tofu have been investigated by many studies, with limited perspectives on the utilization of organic acid coagulants. Hence, this research aimed to study the psychochemical and functional properties of tofu coagulated by bilimbi (Averrhoa bilimbi) and lime (Citrus aurantifolia) juices. The highest tofu protein content was quantified for tofu prepared with 20% bilimbi and 5% lime juices, as much as 52.11 and 52.03% (DW), respectively. The corresponding yield was 73.46 and 69.17%. The hardness (155.98 g), gumminess (116.45 g), and chewiness (112.69 g) of treated tofu were found significantly higher than commercial tofu (p < 0.05). Total phenolic content of commercial tofu was about 370.09 µg GAE/g sample (DW). This value was significantly higher than that of treated tofu (p < 0.05). However, the antioxidant activity of the commercial tofu was lower than tofu coagulated with fruit juices. Based on SDS-PAGE analysis, protein band for 11S fraction of tofu coagulated by bilimbi and lime juices were thicker than that of commercial tofu. These small molecular weight peptides might contribute for higher antioxidant activities of tofu coagulated by bilimbi and lime juices. Conclusively, bilimbi and lime juices are potent natural acid coagulants for enhancing the physicochemical and functional properties of tofu.

Lipase-Catalyzed Interesterification for the Synthesis of Medium-Long-Medium (MLM) Structured Lipids - A Review.

Medium-long-medium (MLM) structured lipids typically contain medium-chain fatty acids (C6-C12) at sn-1,3 and long-chain fatty acids (C14-C24) at sn-2 positions. They have reduced calories and are suitable for the control of obesity, lipid malabsorption and other metabolic disorders. This review focuses on the synthesis of MLM lipids by the enzymatic interesterification. It gives detailed description of biocatalysts, substrates, reactors and synthesis methods, and discusses the use of MLM lipids in food products. The information provided in this review can be considered as the current state-of-the art for developing a future strategy for the synthesis of MLM structured lipids.

Recent advances on prebiotic lactulose production.

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic. The production of lactulose is important in the dairy industry, as it is regarded as a high value-added derivative of whey or lactose. The industrial production of lactulose is still mainly done by chemical isomerization. Due to concerns on the environmental and tedious separation processes, the enzymatic-based lactulose synthesis has been regarded as an interesting alternative. This work aims at comparing chemical and enzyme-catalyzed lactulose synthesis. With an emphasis on the latter one, this review discusses the influences of the critical operating conditions and the suited operation mode on the transgalactosylation of lactulose using microbial enzymes. As an update and supplement to other previous reviews, this work also summarizes the recent reports that highlighted the enzymatic isomerization of lactose using cellobiose 2-epimerase to produce lactulose at elevated yields.

NMR fingerprinting of depolymerised durian rind pectin and its antibacterial activity.

This study investigated the effect of commercial sterilisation treatment (121 °C for 15 min) on durian rind pectin. The change of structure was observed by NMR (nuclear magnetic resonance) and the antibacterial activity was assessed by microdilution method to obtain the minimum inhibitory concentration (MIC) value. NMR spectra revealed the rhamnogalacturonan-I and homogalacturonan structure, with lower methyl-ester in sterilised pectin. Native pectin was unable to inhibit Escherichia coli and Staphylococcus aureus at the highest tested concentration (MIC > 25 mg/mL), but sterilised pectin showed inhibitory effect against E.coli (MIC 12.5 mg/mL) and S. aureus (MIC 6.25 mg/mL). Membrane filtration to obtain fraction < 20 kDa enhanced the inhibition against S. aureus further, but not for E.coli. The antibacterial effect was possibly correlated to the decrease of molecular weight (MW) and degree of esterification (DE) of durian rind pectin. E. coli was more resistant to pectin than S. aureus.

Membrane-based preparative methods and bioactivities mapping of tempe-based peptides.

This study was aimed to produce bioactive peptides from optimally fermented tempe, and map their overall bioactivities. There were three preparative methods utilized for producing tempe-based peptides, such as water-facilitated extraction, alcalase, and papain hydrolysis, and in combination with membrane filtration. Fermenting soybean at 144 h was selected as the optimum time based on protein content and degree of hydrolysis. Through SDS-PAGE analysis, an increased degree of hydrolysis with longer fermentation time was confirmed. The best preparative method for producing bioactive peptides was through papain hydrolysis and followed by 5 kDa membrane filtration. By this, the enhancement was distinct for antioxidant activity, ACE-, α-glucosidase-, and Kunitz trypsin-inhibitory activity. The annotated peptide sequences resulting from Nano LC Ultimate 3000 Series System tandem Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer were matched with the BIOPEP database. The major bioactivities of tempe peptides obtained were as an ACE inhibitor, antioxidant, and antithrombotic.

Influences of operating conditions on continuous lactulose synthesis in an enzymatic membrane reactor system: A basis prior to long-term operation.

Lactulose synthesis was performed in a continuous stirred enzymatic membrane reactor. Each investigated operating condition (agitation, pH, feed molar ratio of lactose to fructose (mL/mF ratio), hydraulic residence time (HRT)) had an influence on reaction performances, in terms of lactulose concentration, productivity and selectivity. Lactulose concentration was maximum at an mL/mF ratio of 1/2. Higher than this ratio, synthesis of galactooligosaccharides was promoted rather than lactulose. At mL/mF ratios lower than 1/2, enzyme inhibition was pronounced to the detriment of lactulose production. At 7 or 9h HRT, higher lactulose concentrations were obtained than at shorter HRTs. Applying an mL/mF ratio of 1/2 and an HRT of 9h in a long-term operation, nearly constant lactulose concentration was reached after 23h and lasted up to 32h with a mean concentration of 14.51±0.07g/L and a reaction selectivity of 0.075-0.080mollactulose/molcons.lactose. After 7d, lactulose concentration reduced by 31%. A continuous synthesis of lactulose at lab-scale was shown to be amenable using a membrane reactor process. Moreover, for process evaluation, this study can bridge the gap between batch laboratory scale and continuous full-scale operation regarding lactulose synthesis.

Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis.

Newly developed parallel small-scale enzymatic membrane reactors (EMRs) were used to enhance the synthesis of lactulose using ß-galactosidase. Under batch operation, the productivity of lactulose decreased abruptly from 2.72 down to 0.04 mg lactulose/(Uenzymeh) over 35 h of reaction. This was presumably caused by the action of ß-galactosidase which performed secondary hydrolysis upon the produced lactulose. The continuous operations of an EMR system led to continuous removal of lactulose in the reactors restricting lactulose degradation caused by secondary hydrolysis. Therefore, continuous lactulose syntheses in the EMRs yielded significantly higher specific productivities under "steady state" conditions. Approximately 0.70 and 0.50 mg lactulose/(U enzyme h) for hydraulic residence times of 5 and 7h were reached, respectively. Continuous lactulose synthesis performed in an EMR system conclusively can circumvent the drawbacks (e.g., secondary hydrolysis) of lactulose synthesis encountered in batch operation. It is, therefore, beneficial in terms of enhanced lactulose productivity and reduced enzyme consumption.

Effect of pellet size and stimulating factor on the glucosamine production using Aspergillus sp. BCRC 31742.

The higher GlcN production using a wild-type fungi, Aspergillus sp. BCRC 31742 cultivated under submerged fermentation was investigated. Several fermentation aspects were studied, such as pellet size, working volume, agitation rate and stimulating factor. Culture cultivation with conditions, such as pellet diameter of 2.15mm, 50mL working volume (250mL T-flask), incubation at 30 degrees C, 200rpm and pH 7.0 for 5days yielded highest biomass concentration which was 33.82g/L, with a GlcN concentration of 7.05g/L. Methanol was found to give the best stimulatory effect in terms of GlcN concentration as compared to glutamic acid, cycloheximide and ethanol. Addition of methanol (1.5%v/v) into fermentation medium could increase GlcN content from 0.21 (control) to 0.26g/gdw cells and led to maximum GlcN concentration of 7.48g/L obtained.