Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg⁻¹ and 12.51 mg mL⁻¹ protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g⁻¹ cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).     

Aqueous medium enzymatic preparation of <Emphasis Type="SmallCaps">l</Emphasis>-alpha glycerylphosphorylcholine optimized by response surface methodology

The ability of Rhizopus chinensis lipase (Thermo 4S-3) to catalyze the deacylation of l,2-diacyl-sn-glycero-3-phosphocholines (sn-1,2-PC) for l-alpha glycerylphosphorylcholine (l-α-GPC) preparation was investigated. Response surface methodology (RSM), based on a modified central composite rotatable design, was employed to examine the effects of substrate concentrations, temperature, enzyme loading, and dosage of CaCl₂ on the l-α-GPC yield. RSM analysis indicated good correlation between experimental and predicated values. The optimal condition was confirmed as follows: reaction time, 3.5 h; temperature, 43 °C; enzyme loading, 28.2 U mL⁻¹; substrate concentration, 51.5 mg mL⁻¹; and dosage of CaCl₂, 1.9 mg mL⁻¹. Under these conditions, the l-α-GPC yield increased by 96.8%, which was close to the amount predicted by the model.     

Determination and Evaluation of the Mineral Composition of Chinese Cabbage (Beta vulgaris)

Inductively coupled plasma optical emission spectrometry was used to determine the elements present in Chinese cabbage (Beta vulgaris). The accuracy of the method was confirmed by analysis of a certified reference material of spinach leaves. The study involved 57 samples that were collected in 13 Brazilian cities. Average concentrations of elements found per gram of Chinese cabbage were as follows: 3.44 mg g⁻¹ sodium, 5.09 mg g⁻¹ potassium, 1.25 mg g⁻¹ phosphorous, 0.85 mg g⁻¹ calcium, 0.49 mg g⁻¹ magnesium, 2.79 μg g⁻¹ manganese, 9.50 μg g⁻¹ iron, 0.74 μg g⁻¹ copper, 14.28 μg g⁻¹ zinc, and 6.44 μg g⁻¹ strontium. Principal component analysis and hierarchical cluster analysis demonstrated that there is no systematic difference in the mineral composition between the cabbage samples that were analyzed.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

An approach to improve ACE-inhibitory activity of casein hydrolysates with plastein reaction catalyzed by Alcalase

The preparation method of casein hydrolysates with high ACE-inhibitory activity was studied by Alcalase-catalyzed hydrolysis coupled with plastein reaction. Casein hydrolysates with an IC₅₀ value of about 47 μg mL⁻¹ were first prepared by hydrolysis of casein with Alcalase and then modified with plastein reaction catalyzed by the same enzyme. The impacts of four reaction conditions on plastein reaction of casein hydrolysates were studied, and then optimal conditions were determined using response surface methodology with the decrease of free amino groups in the reaction mixture as response. When the concentration of casein hydrolysates was fixed at 35% by weight, the maximum decrease of free amino groups in the reaction mixture of 181.8 μmol g⁻¹ proteins was obtained. The optimum conditions for the above decrease were found to be an E/S ratio of 7.7 kU g⁻¹ proteins, reaction temperature of 42.7 °C and reaction time of 6 h. Analysis results showed that ACE-inhibitory activity of casein hydrolysates prepared could be improved significantly by plastein reaction. When casein hydrolysates were modified by plastein reaction, with a decrease of free amino groups in the mixture of about 154.7 μmol g⁻¹ proteins and 181.8 μmol g⁻¹ proteins, their IC₅₀ values could be decreased to 0.6 and 0.5 μg mL⁻¹.     

Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese production

The optimization of culture medium composition was carried out for improvement the recombinant cyprosin B production, an enzyme with high milk-clotting activity. Response surface methodology (RSM) was applied to evaluate the effect of variables namely glucose, yeast extract (YE) and bactopeptone present in the culture medium, used for recombinant cyprosin B production by transformed Saccharomyces cerevisiae BJ1991 strain in shake-flask and bioreactor culture conditions. The central composite experimental design (CCD) was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The optimal concentration estimated for each variable related to a theoretical maximum of cyprosin B activity (488 U mL⁻¹) was 30 g L⁻¹ glucose, 15 g L⁻¹ YE and 27 g L⁻¹ bactopeptone. The optimized medium composition, based on empirical model, led to a cyprosin B activity of 519 U mL⁻¹, which corresponds to an increase of 46%. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in biomass (82%) and recombinant cyprosin B production (139%). The improvement in the recombinant cyprosin B production after optimization process can be considered adequate for large-scale applications, and the clotting activity of cyprosin B account for their use in industrial cheese making.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Effect of dynamic high-pressure microfluidization at different temperatures on the antigenic response of bovine β-lactoglobulin

The antigenic response of β-lactoglobulin (β-Lg), treated by dynamic high-pressure microfluidization (DHPM) at different temperatures, was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from rabbit serum. DHPM treatment causes changes in the protein structure and may influence the antigenicity of β-Lg. DHPM treatment of β-Lg at 90 °C showed significant effects with the antigenic response of 5.2 μg mL⁻¹ (untreated), 45 μg mL⁻¹ (40 MPa), 79 μg mL⁻¹ (80 MPa), 132 μg mL⁻¹ (120 MPa), and 158 μg mL⁻¹ (160 MPa). In combination with temperature treatment (70–90 °C), the antigenic response enhanced as the temperature increased at 160 MPa. The β-Lg antigenicities were about 14, 108, and 158 μg mL⁻¹ at 70, 80, and 90 °C, respectively. However, the influence of DHPM pressures on the antigenic response of β-Lg standards was different. DHPM modified β-Lg standards showed a remarkable increase in antigenicity when treated to 80 MPa. Above 80 MPa, the antigenic response decreased.     

Optimization and Modeling of Process Parameters for Lipase Production by Bacillus brevis

Statistical evaluation of fermentation conditions and nutritional factors by Plackett–Burman two-level factorial design followed by optimization of significant parameters using response surface methodology for lipase production by Bacillus brevis was performed in submerged batch fermentation. Temperature, glucose, and olive oil were found to be the significant factors affecting lipase production. Maximum lipase activity of 5.1 U ml⁻¹ and cell mass of 1.82 g l⁻¹ at 32 h were obtained at the optimized conditions of temperature, 33.7 °C; initial pH, 8; and speed of agitation, 100 rpm, with the medium components: olive oil, 13.73 ml l⁻¹; glucose, 13.98 g l⁻¹; peptone, 2 g l⁻¹; Tween 80, 5 ml l⁻¹; NaCl, 5 g l⁻¹; CH₃COONa, 5 g l⁻¹; KCl, 2 g l⁻¹; CaCl₂·2H₂O, 1 g l⁻¹; MnSO₄·H₂O, 0.5 g l⁻¹; FeSO₄·7H₂O, 0.1 g l⁻¹; and MgSO₄·7H₂O, 0.01 g l⁻¹. The lipase productivity and specific lipase activity were found to be 0.106 U (ml h)⁻¹ and 2.55 U mg⁻¹, respectively. Unstructured kinetic models and artificial neural network models were used to describe the lipase fermentation. The kinetic analysis of the lipase fermentation by B. brevis shows that lipase is a growth-associated product.     

Antioxidant capacity and antioxidative compounds in barley (<Emphasis Type="Italic">Hordeum</Emphasis><Emphasis Type="Italic">vulgare</Emphasis> L.) grain optimized using response surface methodology in hot air roasting

Response surface methodology was used to predict optimum conditions for hot air roasting of barley grains (temperature, time, and amount). Antioxidant capacity in the grains was highest under optimum conditions of 250 °C, 63.5 min and 42 g (one and a half layers). A correlation of R ² = 0.74 (p < 0.05) was found between 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic contents. Ethanol and aqueous extracts were prepared from grains roasted under optimum conditions and assessed for antioxidant capacity. Antioxidative compounds in the extracts were then identified using GC–MS. The IC₅₀ value of ethanol extract was significantly lower (11.45 μg mL^–1) than that of aqueous extract (33.54 μg mL^–1) and α-tocopherol (12.6 μg mL^–1) but higher than BHT (9.59 μg mL^–1). The same trend was observed in linoleic acid assay. In reducing power, the ethanol extract and α-tocopherol were not significantly different. Phenolic acids p-hydroxybenzaldehyde, vallinic and gallic acids were identified as the major compounds in the extracts. The results obtained from this study show that it is possible to optimize antioxidant capacity in barley grains during roasting.     

Determination of Melamine in Liquid Milk and Milk Powder by Titania-Based Ligand-Exchange Hydrophilic Interaction Liquid Chromatography

A simple and rapid high-performance liquid chromatography (HPLC) procedure for the analysis of melamine in liquid milk and milk powder has been developed. Decrease of acetonitrile percentage and phosphate buffer concentration in mobile phase, and lowering of buffer pH and column temperature would benefit the retention of melamine on titania. Taking advantage of the ligand-exchange and hydrophilic interaction mixed retention mode on bare titania column, neither complex pretreatment nor ion-pair reagent was required. The whole analysis for one sample including sample pretreatment and HPLC analysis could be accomplished within 30 min. The method presented good linearity (R ² = 0.9998) in a wide range of 0.02–10 μg mL⁻¹. The limit of detection (3σ) and limit of quantification (10σ) of the method were 6 and 20 μg L⁻¹, respectively, which were equivalent to 15 and 50 μg kg⁻¹ melamine in liquid milk, 60 and 200 μg kg⁻¹ melamine in milk powder, respectively. Such sensitivity could be compared with those obtained by HPLC with solid-phase extraction or HPLC coupled with tandem mass spectrometry and was adequate for the screening of melamine in tainted dairy products. The repeatability (RSDs) of the retention times and peak areas of 11 replicate detections of 1.0 μg mL⁻¹ melamine were 0.32% and 2.5%, respectively. The intermediate precision on three consecutive days (RSDs, n = 6) of the retention times and peak areas were 1.1% and 2.3%, respectively. The recovery of spiked melamine in dairy samples ranged from 95.2% to 105%. The simplicity, sensitivity and rapidity of the proposed method make it an effective alternative detecting technique for melamine.     

Polycyclic aromatic hydrocarbons (PAHs) in traditional and industrial smoked beef and pork ham from Serbia

Smoked beef and pork ham samples were analysed during process of smoking (after packing and storing) for the presence of the 16 EU priority PAHs via Fast GC/HRMS method. This study showed that there are differences in PAH contents between final smoked beef ham samples from traditional smokehouse (TS) (3.9 μg kg⁻¹) and industrial smokehouse (IS), (1.9 μg kg⁻¹). Also there is a difference in PAH contents in final smoked pork ham samples (4.9 μg kg⁻¹, TS; 4.2 μg kg⁻¹, IS). In beef and pork ham samples from the same smokehouse different PAH contents were observed during smoking. The highest content of examined PAHs in all beef and pork ham samples during smoking showed benzo[c]fluorene (BcL) (beef ham: from 0.3 μg kg⁻¹ to 1.5 μg kg⁻¹; pork ham: from 0.2 μg kg⁻¹ to 2.1 μg kg⁻¹).The maximum level for benzo[a]pyrene (BaP) of 5 μg kg⁻¹ in smoked meat products was not exceeded in any samples. Correlation statistic analysis (P < 0.05) of obtained contents from samples both from TS and IS showed that BaP is a good marker both for 16 EU priority PAHs and 12 IARC probably and possibly carcinogenic PAHs (IS: R _BaP/Σ16PAHs = 0.95, R _BaP/Σ12PAHs = 0.96; TS: R _BaP/Σ16PAHs = 0.71, R _BaP/Σ12PAHs = 0.88).     

Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

Effects of Maillard reaction conditions on the antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with maltose

The effects of Maillard reaction conditions (weight ratio of protein to sugar, temperature and time) on the antigenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in conjugates of whey protein isolate (WPI) with maltose were investigated. Response surface methodology was used to establish models to predict the antigenicity of α-LA and β-LG and find an optimal reaction condition under which the antigenicity of α-LA and β-LG reduces to minimum value. Conjugating WPI with maltose was an effective way to reduce the antigenicity of α-LA and β-LG. The antigenicity of α-LA decreased from 32.25 μg mL⁻¹ to 10.91 μg mL⁻¹. And the antigenicity of β-LG decreased from 272.4 μg mL⁻¹ to 38.17 μg mL⁻¹. Temperature had the greatest effect on the antigenicity of α-LA, while weight ratio of WPI to maltose was the most significant factor on the antigenicity of β-LG.     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

Immobilization of <Emphasis Type="BoldItalic">Aspergillus oryzae</Emphasis> β-Galactosidase onto Duolite A568 Resin via Simple Adsorption Mechanism

In this study, a rapid, simple and economic method of enzyme immobilization was developed to hydrolyze lactose. Duolite A568 resin was used for the immobilization of β-galactosidase via simple adsorption mechanism. The effects of immobilization parameters such as time, pH, and temperature were studied. Immobilization parameters for maximum enzyme activity were estimated at 35 °C temperature, pH 4.5, 5 mg/mL enzyme concentration, and approximately 60 min immobilization time. A significant amount of enzyme was immobilized with high catalytic activity. Enzyme immobilization procedure explained in this study slightly affected the enzyme kinetic. The value of Michaelis constant K _m for immobilized enzyme was significantly larger, indicating decreased affinity by the enzyme for its substrate. It was observed that both free and immobilized enzyme showed maximum activity at 65 °C reaction temperature. Immobilized β-galactosidase was significantly more active at all temperatures as compared to its free form. However, optimal pH of immobilized enzyme was slightly affected by immobilization procedure. The optimum pH of immobilized enzyme was shifted up 0.5 unit to a more alkaline value of 6.0 compared to the free enzyme.     

Analytical procedure based on slurry sampling for determining selenium in organic vegetable samples by graphite furnace atomic absorption spectrometry

This paper presents a simple, fast and sensitive method to determine selenium in vegetable samples by graphite furnace atomic absorption spectrometry through the direct introduction of slurries of the samples into the spectrometer’s graphite tube. The limits of detection and quantification calculated for 20 readings of the blank of the standard slurries (5 mg mL⁻¹ of microcrystalline cellulose) were 0.33 and 1.10 μg L⁻¹. The proposed method was applied to determine selenium in samples of organically grown vegetables and its results proved compatible with those obtained from samples mineralized by acid digestion in a microwave oven.     

ITS-RFLP characterization of black <Emphasis Type="Italic">Aspergillus</Emphasis> isolates responsible for ochratoxin A contamination in cocoa beans

In the present study, ochratoxigenic mycobiota in cocoa beans was identified at species level by digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI. Of the 132 isolates of Aspergillus section Nigri collected from cocoa beans, 89 were identified as A. tubingensis, 27 as A. niger, 10 as A. tubingensis-like and 6 as A. carbonarius. No variation was observed between RFLP patterns (C, N, T1 and T2) described previously for grape isolates and those of the cocoa isolates analysed. With respect to OTA-producing fungi, a high percentage of black aspergilli (50.7%) was able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 μg g⁻¹ to 100 μg g⁻¹. Percentages of OTA-producing isolates in the A. niger aggregate were higher than in other substrates, ranging from 30% to 51.7%. Furthermore, the detected levels of OTA production in the A. niger aggregate, particularly in A. tubingensis species was higher than in A. carbonarius, ranging from 0.7 μg g⁻¹ to 120 μg g⁻¹ (mean 24.55 μg g⁻¹). Due to the high occurrence, percentage of ocratoxigenic isolates and their ability to produce OTA, isolates belonging to the A. niger aggregate could be considered as the main cause of OTA contamination in cocoa beans used for manufacturing cocoa products.     

Lactulose production from lactose by recombinant cellobiose 2‐epimerase in permeabilised Escherichia coli cells

Caldicellulosiruptor saccharolyticus cellobiose 2‐epimerase (CsCE) catalyses the single substrate lactose into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. Ethanol‐permeabilised Escherichia coli cells containing CsCE were used as biocatalysts for lactulose production. The reaction conditions for maximum lactulose production were optimised to be 600 g L^?1 lactose, pH 7.5, 80 °C and 12.5 U mL^?1 of whole‐cell biocatalyst. After incubated at Na₂HPO₄–NaH₂PO₄ buffer for 2 h, approximately 390.59 g L^?1 lactulose was obtained with a conversion yield of 65.1%. The lowest production and conversion yield of epilactose were also found in Na₂HPO₄–NaH₂PO₄ buffer with a final concentration of 11.7 g L^?1 and a conversion yield <2%. The results represent a promising technology to attain high production and conversion yield of lactulose with a high purity on industrial scale.