Chaperone‐like activity of β‐casein and its effect on residual in vitro activity of horseradish peroxidase

In this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone‐like activity of β‐casein under elevated temperature. It was shown that β‐casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between proteins. Incubating HRP (0.1 mg mL^?1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL^?1 β‐casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β‐casein on HRP disappears when pH is below the isoelectric point of β‐casein. It was also proven by light scattering studies that β‐casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries such as enzyme manufacturers and food industry.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Comparison of releasing bound phenolic acids from wheat bran by fermentation of three Aspergillus species

Wheat bran was fermented at 28 °C for 7 days under 70% humidity by Aspergillus niger, Aspergillus oryzae and Aspergillus awamori. Total phenolic content (TPC) of the unfermented sample was 1531.5 μg g^?1 wheat bran. After the fermentation of Aspergillus awamori, Aspergillus oryzae and Aspergillus niger, TPC reached 5362.1, 7462.6 and 10 707.5 μg g^?1, respectively. The antioxidant activity in the extractions of fermented wheat bran also increased significantly compared with the unfermented sample (P < 0.05). Aspergillus niger showed the greatest capacity to release bound ferulic acid (416.6 μg g^?1). Aspergillus oryzae and Aspergillus awamori had the advantages of releasing more chlorogenic acid (84.0 μg g^?1) and syringic acid (142.3 μg g^?1), respectively. The destructive effect of Aspergillus niger on wheat bran structure was the strongest, followed by that of Aspergillus oryzae. This effect of Aspergillus niger may be due to its higher cellulase, xylanase, arabinofuranosidase and β‐xylosidase activities. Besides, Aspergillus oryzae possessed higher β‐glucosidase activity, and Aspergillus awamori had higher α‐amylase and feruloyl esterase activities. Aspergillus niger may be the best to release bound phenolic acids in the three Aspergillus species. These will provide the helpful information for understanding mechanism of the fermentation by Aspergillus species releasing bound phenolic in wheat bran.     

Preparation and purification of angiotensin‐converting enzyme inhibitory peptides from hydrolysate of shrimp (Litopenaeus vannamei) shell waste

Angiotensin I‐converting enzyme (ACE) inhibitory peptides from the shrimp shell waste (SSW) were isolated using different proteases. The orthogonal test results showed alcalase hydrolysates with ACE inhibitory activity of 67.07% under the optimal hydrolysis conditions of 60 °C hydrolysis temperature, pH = 9.5, 25 g L^?1 substrate and 4000 U g^?1 of enzyme, whereas neutral protease hydrolysates had an ACE inhibitory activity of 84.04% under the hydrolysis temperature of 50 °C at pH = 7.0 with 25 g L^?1 of substrate and in the presence of 2000 U g^?1 of enzyme. Neutral protease was more suitable for the production of ACE inhibitory peptides from SSW, where peptides with MW <5 kDa were recommended. The results of this study indicated that peptides obtained from SSW are as beneficial as antihypertension compounds in the functional food resources.     

Production optimization,purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 × 10⁵ g^?1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg^?1. Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn²⁺ enhanced its activity while Co²⁺ showed inhibitory effect. With casein as substrate, the kinetic parameters of K _m, V _max, energy of activation (E _a), and attenuation index of inactivation velocity by heat inducing (λ) were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1, and 0.59, respectively.     

Effect of gums on viability and β‐galactosidase activity of Lactobacillus spp. in milk drink during refrigerated storage

The viability and β‐galactosidase activity of four Lactobacillus strains in milk drink containing gums during 28 days of refrigerated storage at 4 °C were assessed. The population of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were maintained, whereas the population of Lactobacillus reuteri DSM20016 and Lactobacillus reuteri SD2112 significantly decreased. The recommended level of 6 log CFU g^?1 was exceeded for all tested trains throughout storage. The highest viable number of Lactobacillus rhamnosus GGB103 (8.76 ± 0.03 log CFU mL^?1) was obtained in the product containing carrageenan–maltodextrin. The addition of guar–locust bean–carrageenan led to 20‐fold increase in the level of β‐galactosidase activity for L. rhamnosus GGB101 (1208 ± 2.12 Miller units mL^?1) compared to the control (61 ± 2.83 Miller units mL^?1). Our results suggested that gums could be added to milk to improve viability and enhance β‐galactosidase activity of Lactobacillus.     

Interactions of digestive enzymes and milk proteins with tea catechins at gastric and intestinal pH

The interactions of digestive enzymes (pepsin, pancreatin) and milk proteins (β‐casein, β‐lactoglobulin (β‐Lg)) with (?)‐epigallocatechin gallate (EGCG), (?)‐epigallocatechin (EGC) and (?)‐epicatechin (EC) at gastric and intestinal pH were investigated by fluorescence spectroscopy. The results indicated that in the gastric environment, all three tea catechins showed binding affinities in descending order of strength with β‐casein first, followed by β‐Lg and then pepsin. The highest affinity was observed for EGCG–β‐casein, with a binding constant (K_A) of 2.502(±0.201) × 10⁵ m ^?1. In the intestinal environment, the binding strengths of the proteins with EGCG and EGC were in the order β‐Lg > pancreatin > β‐casein; for binding with EC, the strength order was β‐casein > β‐Lg > pancreatin. The combination EGCG–β‐Lg had the strongest binding affinity, with a K_A of 14.300(±0.997) × 10⁵ m ^?1. Thermodynamic analysis revealed that tea catechins complexed with milk proteins and digestive enzymes via different hydrophilic and hydrophobic interactions depending on the different digestion environments and types of catechins, proteins and enzymes.     

Purification and characterisation of soluble acid invertase from mango fruits

Soluble acid invertase (SAI) was purified from mango fruits (Mangifera indica L.) by ammonium sulphate fractionation and anion‐exchange chromatography (DEAE‐Sepharose Fast Flow). Molecular mass of the enzyme is 45 kDa estimated by SDS–PAGE. Dynamic light scattering analysis suggests the hydrodynamic radius of SAI distributes from 4 to 20 nm with a peak at 6.68 nm. Transmission electron microscopy shows that SAI is a globulin with diameter of 10–30 nm. Its optimal pH and temperature are 4.0 and 60 °C, respectively. The enzyme is not stable at high temperature (≥60 °C) or in alkaline (pH ≥ 8) environment. Using sucrose as substrate, its K_M and V_max are 25.55 mm and 1.002 mmol min^?1 mL^?1, respectively. Its circular dichroism spectrum shows a negative band at 220 nm and a positive band at 195 nm, suggesting a β‐sheet structure. The fluorescence spectra reflect that the tryptophan and tyrosine residues of SAI are partially exposed.     

Breaking of structural dormancy in seeds of Tetrapleura tetraptera by Aspergillus niger

Aspergillus niger (strain UUF9202) significantly reduced the dormancy period and boosted aggregate germination percentage (AGP) in seeds of Tetrapleura tetraptera. A net reduction in mean germination time (MGT) to 12 days and an increase from 0 to 42% AGP were obtained in 32 days. The optimal spore inoculum of the fungus was 8.5 × 10≥10 ml^?1 per 100 seeds in 96 h pregermination incubation. The potential of some physical factors in combination with this fungal concentration in enhancing AGP was evaluated. The treatments were as follows: (i) hot water scarification at 30°C, 50°C, 80°C and 100°C plus A niger; and (ii) nicking by decoating at either the micropylar or chalazal ends plus A niger. Micropylar scarification plus A niger gave the highest AGP (94%) followed by chalazal scarification plus A niger (86%) and hot water dip at 80°C + A niger (82%). The control seeds which were soaked in presterilised (by autoclaving at 121°C for 15 min) A niger spore suspension at 30°C gave zero germination.     

Preparation and characteristics of squid pen β‐chitin prepared under optimal deproteinisation and demineralisation condition

Squid (Todarodes pacifica) pen was an excellent source of β‐chitin with 25.5% yield. The optimal condition to prepare squid pen β‐chitin was established: deproteinisation with 3% NaOH for 30 min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v) and a subsequent demineralisation with 1 N HCl for 30 min at room temperature and a solid/solvent ratio of 1:10 (w/v). Squid pen β‐chitin contained 6.29% nitrogen, 0.25% ash, and negligible fat with degree of acetylation of 94.02%, residual amino acid of 0.499 g/100 g and bulk density of 0.28 g mL^?1. Depending on its particle size, squid pen β‐chitin visually looked white (L* = 82.82, a* = ?0.67, b* = 6.31; particle size of 0.15–0.18 mm) or light grey (L* = 62.88, a* = 0.33, b* = 10.66; particle size of 0.425–0.841 mm). Water, fat and dye‐binding capacity of squid pen β‐chitin was 694.67%, 194.03% and 79.81%, respectively.     

Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains

In this study, we investigated the potential of aflatoxin B₁ (AFB1) production by five Aspergillus flavus strains previously isolated from sorghum grains on cereals (barley, maize, rice, wheat and sorghum), oilseeds (peanuts and sesame) and pulses (greengram and horsegram). Five strains of A. flavus were inoculated on all food grains and incubated at 25 °C for 7 days; AFB1 was extracted and estimated by enzyme‐linked immunosorbent assay. All A. flavus strains produced AFB1 on all food grains ranging from 245.4 to 15 645.2 μg kg^?1. Of the five strains tested, strain Af 003 produced the highest amount of AFB1 on all commodities ranging from 2245.2 to 15 645.2 μg kg^?1. Comparatively, the AFB1 accumulation was high on rice grains ranging from 3125.2 to 15 645.2 μg kg^?1, followed by peanuts ranging from 2206.2 to 12 466.5 μg kg^?1. Less AFB1 accumulation was observed in greengram and sesame seeds ranging from 645.8 to 2245.2 and 245.4 to 2890.6 μg kg^?1, respectively. Our results showed that all food grains tested are susceptible to A. flavus growth and subsequent AFB1 production.     

Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Composition,characterisation and analysis of seed oil of Suaeda salsa L

Awareness of vegetable oils being beneficial for health has attracted researchers in exploring different vegetable oils. In this study, Suaeda salsa L. seed oil was extracted and characterised. The yield of S. salsa L. seed oil was 25.99%. Acidity, iodine number, saponification number, peroxide value and unsaponifiable matter were used to assess seed oil quality. Melting point and melting enthalpy were found to be ?35.75 °C and 26.39 J g^?1, respectively, from differential scanning calorimeter‐melting curves. Fatty acid and triacylglycerol composition of this oil was analysed by GC‐MS and HPLC‐MS, respectively. The main fatty acid in S. salsa L. seed oil is Linoleic (65.03%), and the dominant triacylglycerols (TAGs) were LLL and OLL. The oil was found to have high amounts of α‐tocopherol (36.72 mg 100 g^?1) and β‐tocopherol (34.76 mg 100 g^?1). All the results suggest that S. salsa L. seed oil may have potential applications in relating industries.     

Characterization of amylase and protease produced by Aspergillus awamori in a single bioreactor

The aim of this study was to characterize the glucoamylase and acid protease produced in a single bioreactor by Aspergillus awamori: nakazawa MTCC 6652. Both the enzymes were found stable in wide range of pH (3–9) and temperature (25–70 °C). Optimum activities of amylase and protease were obtained at pH 4 and 5, respectively, whereas 70 and 55 °C had been found as most suitable temperature for highest activities of amylase and protease, respectively. Half life of glucoamylase was 210, 120, 60 and 35 min at 50, 60, 70 and 80 °C, respectively, which was 150, 120, 65 and 15 min at 40, 50, 60 and 70 °C, respectively, for acid protease. K_m and V_max of glucoamylase and protease were 9.8 mg/ml, 56.2 mg/ml/min and 1.08 mg/ml, 8.8 mg/ml/min, respectively. In low amount (1 mM) almost all metal ions except Mn, such as Ca²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, Zn²⁺ and Hg²⁺ enhanced glucoamylase activity whereas protease activity was inhibited by all the ions except Zn²⁺. At low concentration, i.e., (0.03% w/v) Triton X-100 and SLS increased the activity of glucoamylase, while in higher concentration it inhibited activities of both the enzymes. β-mercaptoethanol at 0.25% (v/v) enhanced the amylase and protease activity by 1.6 and 3.0 fold, respectively, whereas the presence of 0.5% (v/v) β-mercaptoethanol inhibited the activities of both the enzymes drastically. At 0.5 M concentration of urea, glucoamylase activity increased but drastic inhibition took place at 5 M urea. In case of protease, 0.5 M of urea enhanced its activity and 1 M urea inhibited it completely. Thus, glucoamylase and protease produced by A. awamori nakazawa confirm their suitability for diverse applications in industries.     

Effects of microwave and hot‐air drying methods on colour, β‐carotene and radical scavenging activity of apricots

The effects of drying by microwave and convective heating at 60 and 70 °C on colour change, degradation of β‐carotene and the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH) scavenging activity of apricots were evaluated. Microwave heating reduced significantly the drying time (up to 25%), if compared with convective one, also owing to the higher temperature reached during the last phase of the process, as monitored by infrared thermography. Colour changes of apricot surface, described with lightness and hue angle, in both drying methods followed a first‐order reaction (0.927 ≤ R² ≤ 0.996). The apricots dried by microwave were less affected by the darkening phenomena. The evolution of β‐carotene in fresh apricots (61.2 ± 5.6 mg kg^?1 d.w.) during the drying highlighted a wider decrease (about 50%) when microwave heating was employed for both the temperatures used. Radical scavenging activity increased (P < 0.05) in all dried samples except for hot‐air dried apricots at 60 °C.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Production and stability of water‐dispersible astaxanthin oleoresin from Phaffia rhodozyma

The process of extracting the astaxanthin oleoresin from pretreated Phaffia rhodozyma cells was optimised using a Box‐Behnken response surface design. Microwaving the cells at 105 W for 1 min followed by ethyl acetate extraction was the best pretreatment, and the optimal extraction conditions were 65 °C for 24 min using a solvent–solid ratio of 19:1. The order of the ability to disperse the astaxanthin oleoresin was propylene glycol> Tween 80 > Tween 20 > α‐cyclodextrin, β‐cyclodextrin. It was determined that the degradation of the colour of the water‐dispersible oleoresin followed a first‐order kinetics model. The greatest stability was observed at pH 4 and at the lowest temperature evaluated (40 °C). The thermal degradation of the pigment occurs in two steps, the first one from 0 to 1.5 h, with an E_aI = 10.31 kJ mol^?1, and the second one from 1.5 to 5 h, with an E_aII = 30.06 kJ mol^?1     

Physicochemical properties of extruded germinated wheat and barley as modified by CO2 injection and difference extrusion conditions

Germinated wheat and barley were extruded with a pilot‐scale corotating twin‐screw extruder under different barrel temperatures (100 and 120 °C), feed moisture content (30 and 35%) and CO₂ injection. The effect of germination, barrel temperature, feed moisture content and CO₂ injection on physical properties, proximate composition, γ‐aminobutyric acid, β‐glucan, phytic acid and protease activity was investigated. Results indicated that the barrel temperature, moisture content and CO₂ injection significantly affected water absorption index (WAI), water‐soluble solid index (WSI), specific length, expansion ratio, bulk density, mechanical properties and colour of all extruded products (P < 0.05). Extrusion process significantly decreased γ‐aminobutyric acid and protease activity in extruded germinated wheat (EGW) and barley (EGB) products (with and without CO₂ injection). However, extrusion process (with and without CO₂ injection) increased β‐glucan in EGB and decreased phytic acid in EGW. The content of proximate composition, γ‐aminobutyric acid, β‐glucan, phytic acid and protease activity was slightly affected by CO₂ injection.     

Experimental study on drying of pear slices in a convective dryer

The experiments were conducted on pear slices with thickness of 5 mm at temperatures of 50, 57, 64 and 71 °C with an air velocity of 2.0 m s^?1. Prior to drying, pear slices were pretreated with citric acid solution (0.5% w/w, 1 min, 20 °C) or blanched in hot water (1 min, 85 °C). Also, the untreated samples were dried as control. The shortest drying time of pear slices was obtained with pretreatment with citric acid solution. It was observed that whole drying process of pear slices took place in a falling rate period. Four mathematical models were tested to fit drying data of pear slices. According to the statistical criteria (R², χ² and RMSE), the Midilli et al. model was found to be the best model to describe the drying behaviour of pear slices. The effective diffusivity of moisture transfer during drying process varied between 8.56 × 10^?11 and 2.25 × 10^?10 m² s^?1, while the activation energy of moisture diffusion in pear slices was found to be 34.95–41.00 kJ mol^?1.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling