Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase

In this study, lactulose synthesis from whey lactose was investigated in batch and continuous systems using immobilized β-galactosidase. In the batch system, the optimal concentration of fructose for lactulose synthesis was 20%, and the effect of galactose, glucose and fructose on β-galactosidase activity was determined for hydrolysis of whey lactose and the transgalactosylation reaction for lactulose synthesis. Galactose and fructose were competitive inhibitors, and glucose acted as a noncompetitive inhibitor. The inhibitory effects of galactose and glucose were stronger in the transgalactosylation reaction than they were in the hydrolysis reaction. In addition, when immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity was retained to the extent of 52.9% after 10 reuses. Lactulose was synthesized continuously in a packed-bed reactor. We synthesized 19.1 g/l lactulose during the continuous flow reaction at a flow rate of 0.5 ml/min.     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Production of citreoviridin by Penicillium citreonigrum strains associated with rice consumption and beriberi cases in the Maranhão State,Brazil

The aim of this study was to determine the levels of Penicillium citreonigrum and citreoviridin present in rice samples from Maranhão State, Brazil, where an outbreak of beriberi was reported and 32 deaths occurred (7% of the notified cases died in 2006). The ability of P. citreonigrum to produce citreoviridin was assessed, and a total of 420 samples of 21 different kinds of rice were collected. Mycobiota isolation and identification, the ability of citreoviridin strains to produce toxin, and the natural occurrence of citreoviridin were established. Rice samples were found to have high fungal counts and showed increasing levels from 2004 to 2007 harvest years. The most frequent genus was Aspergillus followed by Penicillium and Cladosporium. Ten out of eleven strains of P. citreonigrum were able to produce citreoviridin. Three rice samples had levels of citreoviridin ranging from 12 to 96.7 ng g^?1, and two bran samples had levels of 128 and 254 ng g^?1. These samples contaminated with P. citreonigrum and citreoviridin were involved in the beriberi cases from Maranhão State. Monitoring rice for mycotoxins in areas where this substrate is the basic food is crucial to prevent outbreaks like the one reported in this study, to improve management practice, and to diminish exposure risk of humans to these harmful toxins.     

Maximization of Fructooligosaccharides and β-Fructofuranosidase Production by Aspergillus japonicus under Solid-State Fermentation Conditions

The conditions of temperature, moisture content, and inoculum rate able to maximize the production of fructooligosaccharides (FOS) and β-fructofuranosidase (FFase) enzyme by solid-state fermentation were established. Fermentation assays were performed using the support material (coffee silverskin) moistened to 60, 70, or 80 % with a 240-g/l sucrose solution and inoculated with a spore suspension of Aspergillus japonicus to obtain 2?×?10⁵, 2?×?10⁶, or 2?×?10⁷ spores/gram dry material. The fermentation runs were maintained under static conditions at 26, 30, or 34 °C during 20 h. The moisture content did not influence the FOS and FFase production; however, temperature between 26 and 30 °C and inoculum rate of approximately 2?×?10⁷ spores/gram dry material maximized the results (FOS?=?208.8 g/l with productivity of 10.44 g/l h; FFase?=?64.12 units U/ml with productivity of 4.0 U/ml h). These results are considerably higher than those obtained under no optimized fermentation conditions and represent an important contribution for the establishment of a new industrial process for FOS and FFase production.     

Evolution of β-lactoglobulin and α-lactalbumin content during yoghurt fermentation

The evolution of the concentrations of β-lactoglobulin (BLG) and α-lactalbumin (ALAC) was studied during the early stages of yoghurt fermentation by YC 191, a mixed strain culture from Chr Hansen containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Radial immunodiffusion of samples taken at different times indicated that the concentration of both proteins remained constant during fermentation. Electrophoresis performed on 12% polyacrylamide slab gels confirmed the results obtained with radial immunodiffusion. In model experiments, the strains were incubated either separately or in combination with both whey proteins, one by one or together. BLG proteolysis required a longer time than that used during yoghurt fermentation. ALAC was susceptible to proteolysis, especially by Streptococcus thermophilus. Despite evident possession of adequate proteolytic system, the strains used for yoghurt production did not cleave detectable amounts of the whey proteins during yoghurt fermentation.     

Production of γ-aminobutyric acid during fermentation of Gastrodia elata Bl. by co-culture of Lactobacillus brevis GABA 100 with Bifidobacterium bifidum BGN4

Gastrodia elata Bl. (GE) is a traditional herbal medicine used for the prevention of cerebrovascular disease and for the regulation of one’s blood pressure. γ-Aminobutyric acid (GABA) is known to be beneficial for preventing neurological disorders and hypertension. The objective of this study was to develop fermented GE products containing high levels of GABA. The optimal medium conditions for the production of GABA during fermentation were as follows: an initial pH of 6.5, 3% (w/v) l-monosodium glutamate, 10% (w/v) freeze-dried GE powder, and 0.5% (w/v) yeast extract. The production of GABA was further enhanced by the co-culture of Lactobacillus brevis GABA 100 with Bifidobacterium bifidum BGN4. During fermentation, high amounts of organic acids and GABA were produced, while gastrodin, the main polyphenol compound of GE, was completely converted to 4-hydroxybenzylalcohol. The fermented GE product with GABA was successfully produced by optimizing the fermentation conditions.     

Whey protein-derived peptide sensing by enteroendocrine cells compared with osteoblast-like cells: Role of peptide length and peptide composition,focussing on products of β-lactoglobulin hydrolysis

The short-term satiating effect of whey protein intake is higher than that obtained with other dietary proteins. The molecular basis underlying the secretagogue effect of whey proteins on the release of cholecystokinin, a gut hormone involved in appetite suppression, has not been deeply investigated. Using STC-1 enteroendocrine cells we analysed the effects of synthetic peptides and protein hydrolysates. The hypothesis that specific molecular features in terms of short amino acid sequences released after enzymatic digestion may exert a primary role in the cholecystokinin secretagogue activity of dietary proteins was not supported. However, the length of the peptide fragments derived from a protein during enzymatic digestion is relevant to this activity. Regarding the complexities of how food can interact with different cellular systems, our data suggest a novel biological activity of ALPMH, a known bioactive peptide product of β-lactoglobulin digestion, on cell growth and a related signalling pathway in osteoblast-like cells.     

Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85°C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.     

Regulation of virus-induced inflammatory response by β-carotene in RAW264.7 cells

Carotenoids are effective antioxidants, which can quench singlet oxygen, suppress lipid peroxidation, and prevent oxidative damage. Both Pseudorabies virus (PRV) and human Herpes simplex virus (HSV) are DNA viruses, and their pathogenesis and immunobiology are similar. However, PRV does not infect humans. Therefore, PRV was used to infect murine macrophages (RAW264.7 cells), to mimic HSV-induced inflammation. Meanwhile, the influence of β-carotene on PRV-induced inflammation was also investigated. Results indicated that β-carotene inhibited (p < 0.05) NO, IL-1β, IL-6, and MCP-1 production in PRV-infected RAW264.7 cells. β-Carotene also suppressed (p < 0.05) NF-κB (p50 and p65), phosphorylation of extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. It could be concluded that the anti-inflammatory effect of β-carotene is mainly through a suppression of cytokine expression in PRV-induced inflammation, which results from NF-κB inactivation. β-Carotene can be considered a potential anti-inflammatory agent for DNA-virus infection.     

Degradation of α-Lactalbumin and β-Lactoglobulin by Actinidin

Susceptibility of the two major whey proteins, β-lactoglobulin and α-lactalbumin, to enzymatic degradation by actinidin as a function of pH and temperature was examined by a response surface methodology in order to elucidate the enzymatic action of the protease for controlled modification of these whey proteins. Pure whey protein fractions and commercial spray-dried whey were degraded by actinidin. The simultaneous effects of pH and temperature, in a range of 2.3 to 5 and 41 to 58°C respectively, on whey proteins degradation were studied, demonstrating a clear interrelationship between these two variables. With commercial whey, extended proteolysis of both β-lactoglobulin and α-lactalbumin was observed at pH 4.0 and temperature of 41.6°C; after an incubation time of 120 min, a degradation of 43.6% was obtained for the former, and 89.1% for the latter. Assays on pure proteins showed a complete degradation of α-lactalbumin and a 65.3% of degradation for β-lactoglobulin; therefore, the former appeared to be more susceptible to actinidin proteolysis.     

Production of Fungal β-amylase and Amyloglucosidase on Some Nigerian Agricultural Residues

Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Helminthosporium oxysporium, and Penicillium frequestans isolated from common Nigerian agricultural residues like cassava, yam, banana, and plantain peels and brewery spent grains (BSG) were screened for their ability to produce β-amylase and amyloglucosidase using submerged and solid-state cultivation regimens. Enzyme activity (EU) was determined by estimating the amount of reducing sugars produced as a result of the action of crude enzyme solutions on buffered starch solution. Results showed that A. niger liberated the highest level of β-amylase (33.2 EU) on plantain peels medium using the static cultivation method, while it produced the highest amount of amyloglucosidase (29.8 EU) on yam peels substrate with solid-state cultivation regimen. Plantain peels favored β-amylase production more than the other tested wastes supporting production of 33.2 and 9.8 EU of the enzyme in A. niger under solid-state and static cultivation methods, respectively. Yam peels favored liberation of 15.3 EU amyloglucosidase in each of the solid-state and the static cultivation regimens. Production of β-amylase on BSG, yam, banana, and plantain peels was not significantly facilitated by static cultivation method (p > 0.05), while solid-state cultivation regimen favored amyloglucosidase production on cassava peels and BSG (p < 0.05). Optimal production yield of β-amylase and amyloglucosidase were 16.6 and 14.9 EU g⁻¹ on plantain and yam waste media by the mold.     

Interaction of fatty acids with oxidation of cholesterol and β-sitosterol

The present study studied the effect of stearic acid, oleic acid, linoleic acid and α-linolenic acid on oxidation of cholesterol and β-sitosterol. Both cholesterol and β-sitosterol were instable with 85% of cholesterol and β-sitosterol being degraded at 180 °C for 120 min. Cholesterol and β-sitosterol had a similar degradation pattern and produced 7-keto, 7α-hydroxy, 7β-hydroxy, 5,6α-epoxy and 5,6β-epoxy oxidative derivatives, indicating position 7 was the most susceptible site of oxidation. Four fatty acids accelerated the degradation of cholesterol and β-sitosterol for the first 60 min at 180 °C and thereafter their oxidation-promoting effect became weaker or diminished. When the oxidation progressed to 120 min, cholesterol and β-sitosterol in the presence of α-linolenic acid, was less oxidised when compared with the control, suggesting that it might inhibit the oxidation. It was concluded that the surrounding fatty acids affected sterol oxidation in a time-dependent manner and their effect was unlikely related to their degree of unsaturation.     

Effect of fermentation on content,molecule weight distribution and viscosity of β-glucans in oat sourdough

This study investigated the effect of fermentation on the physicochemical properties of β-glucans in oat sourdough. Sourdoughs were produced from oat using homo-fermentative lactic acid bacteria, Lactobacillus plantarum 22134. The contents of total β-glucan and soluble β-glucan, the molecular weight (MW) of β-glucan and the viscosity of the extracted β-glucans were determined at 0, 4, 8, 10 and 12 h of fermentation. The total β-glucan content decreased from 4.89% to 4.23% after 12 h of fermentation. The soluble β-glucan concentration increased from 1.89% to 2.18% and then decreased to 1.97% after 8 h of fermentation. The content of β-glucans with MW > 10⁵ decreased from 0 to 4 h of fermentation, followed by an increase and then a decrease after 8 h. The oat sourdough fermented for 8 h had high viscosity, which could be more beneficial for health and bread texture quality, especially for gluten-free breads.     

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3 μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.