Effects of microwave and ultrasound pretreatments on enzymolysis of milk protein concentrate with different enzymes

Milk protein concentrate was pretreated either by microwave irradiation or by ultrasound before initiation of 3‐h enzymatic hydrolysis. The duration of pretreatment ranged from 1 to 8 min at a power level of 800 W, with the control not being subjected to any pretreatment, and five enzymes (Alcalase, Trypsin, Neutrase, Alkaline Protease and Flavourzyme) were employed. The effects of microwave and ultrasound pretreatments on the kinetics and degree of hydrolysis, protein solubility, bitterness and angiotensin‐converting enzyme inhibitory activity were evaluated. Pretreatments increased the degree of hydrolysis and stabilised the solubility of the hydrolysates but could not significantly reduce bitterness of the hydrolysates The angiotensin‐converting enzyme inhibitory activity of the hydrolysates were improved with 5‐min ultrasound‐pretreated Neutrase hydrolysates giving IC₅₀ value of 0.23 mg mL^?1. Kinetic parameters showed improved catalytic efficiencies. Pretreatments of milk protein concentrates with either microwave or ultrasound significantly improve the bioactivity and functional characteristics of the resulting hydrolysates.     

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics,Reaction Model,and Thermodinamics

The impact of ultrasound waves generated by probe‐type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second‐order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k₂) are 0.009, 0.011, 0.053, and 0.045 min^?1 for untreated EWPs, and those pretreated with heat, probe‐type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (E_a) and enzyme deactivation (E_d) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe‐type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.     

Impact of sonication on slurry shear -thinning of protein from sea cucumber (Apostichopus japonicus): Proteolytic reaction kinetics,thermodynamics, and conformational modification

The present study investigated the effect of slit dual-frequency ultrasonic (SDFU)-shear thinning on the kinetics and thermodynamic mechanism of sea cucumber protein (SCP) substrate enzymatic proteolysis reaction. Acoustic cavitation and mechanical shear force applied reduced the protein particle size, intermolecular forces, surface charge and increased the average kinetic energies of the molecules which resulted in substrate viscosity reduction. The enzyme-driven bonds were thus exposed for easy protease accessibility to enhance the reaction rate (k) and reduce Gibbs-free (∆G) and activation energy (Ea) required. Substrate viscosity had a negative significant correlation (r = −0.865, p < 0.01) with SCP conversion degree. SDFU-pretreatment significantly boosted the reaction rate (k_u) of enzymolysis at 20, 30, 40 and 50 °C by 63.0, 71.24, 37.5 and 37.22% respectively over the control (kc). Energy cost required for substrate conversion into product could be tremendously minimized by SDFU-pretreatment as affirmed by the lower values of enthalpy (∆H), entropy (∆S), and activation energy (E_a) (28.30 ± 0.63kJmol⁻¹, −205.70 ± 1.72 Jmol⁻¹ k⁻¹ and 31.01 ± 0.27 kJmol⁻¹ correspondingly) in comparison to the control. Higher cavitation yield and shear force from dual-frequency ultrasonication (20/68 kHz; sequential and simultaneous) resulted in significant modification in the α-helix (reduction), β-turn (reduction), β-sheet (increase), and random coil (increase) of SCP. The structural alterations shown in the circular dichroism (CD), intrinsic fluorescence spectra, scanning electron microscopy (SEM), surface hydrophobicity, and atomic force microscopy (AFM) of pretreated SCP exhibited the extent of modification caused by cavitation and ultrasonic shear force on enzyme-substrate interaction. Hence, ultrasonic-visco reduction can be applied by industries for enzymatic reaction process enhancement.     

Effect of Pretreatment with Dehulling and Microwaving on the Flavor Characteristics of Cold‐Pressed Rapeseed Oil by GC‐MS‐PCA and Electronic Nose Discrimination

Raw and dehulled rapeseeds were treated with microwave energy (800 W) from 1 to 8 min with 1‐min intervals at a frequency of 2450 MHz to investigate the influence of microwaving and dehulling pretreatment on the flavor characteristics of rapeseed oil extracted by pressing. Headspace solid phase microextraction was used to isolate the volatile compounds of rapeseed oil, which were then identified by gas chromatography‐mass spectrometry analysis. The results indicated that microwave and dehulling pretreatment of rapeseed can significantly influence the kinds and content of volatile compounds. The key flavor compounds in rapeseed oil were oxidized volatiles, heterocyclic compounds, and degradation products of glucosinolates. A pungent compound, 4‐isothiocyanato‐1‐butene, was reduced by 97% in rapeseed treated for 3 min with microwaves energy when compared to the rapeseed oil without any treatment. The pyrazine compounds in the oil appeared after 6 min of microwave pretreatment and give a pleasant roasting flavor when compared to crude oils. Principal component analysis was able to differentiate between oils obtained using 4 pretreatment processes based on volatile compounds and electronic nose. The results showed that dehulling pretreatment could improve the flavor, yet microwaving had a greater effect on the flavor of rapeseed oils.     

Effects of calcium chloride and calcium lactate applications with osmotic pretreatment on physicochemical aspects and consumer acceptances of dried papaya

This study explored the effects of calcium chloride (CaCl₂) and calcium lactate (Ca‐L) pretreatments prior to osmotic dehydration on physicochemical characteristics and consumer acceptance of dried papaya. Three different calcium concentrations [0.5, 1.5 and 2.5% (w/v)] were applied to samples before they were soaked in a 30 °Brix sucrose solution and subsequently dried at 70 °C. The results showed that the dried samples treated with calcium exhibited significantly lower moisture content, water activity, apparent density and shrinkage, while volume was higher when compared to the control. Calcium applications did not obviously influence sugar content of samples and maintained the quality of dried papaya in terms of colour and textural characteristics. Calcium chloride at 2.5% (w/v) was found to particularly impart undesirable bitterness to the dried papaya. In contrast, using 2.5% (w/v) Ca‐L provided the best acceptance scores. Thus, the use of Ca‐L could be an alternative to CaCl₂ in the pretreatment of fruits prior to drying as it offers improved quality.     

The effect of calcium chloride and calcium lactate pretreatment concentration on peach cell integrity after high‐pressure processing

The objective of this study was to determine the effect of calcium chloride and calcium lactate pretreatments at different concentrations (1, 1.5 and 2% w/v) on enzymatic browning of clingstone and freestone peaches following high‐pressure processing (HPP) at 200 MPa for 10 min. Proton nuclear magnetic resonance (¹H‐NMR) relaxometry indicated that following calcium pretreatment and HPP, both peach types had lower percentages of water in the vacuole compartment and a simultaneous increase in the percentage of water in the cytoplasm compartment. Calcium pretreatment of freestone peaches did not affect the development of browning, regardless of the form of calcium or the concentration, whereas calcium lactate pretreatment undesirably enhanced clingstone peach discoloration. Calcium pretreatments have the potential to reduce the loss of cell integrity that results from HPP; however, in this study, the effect was not sufficient to inhibit the interaction between the polyphenol oxidase enzyme that catalyses browning and its substrates.     

Effect of some thermal and chemical pretreatments on smoked Oyster mushroom quality

The effect of different thermal and chemical pretreatments on quality and enzyme activities of smoked mushroom was investigated. Mushrooms were blanched (water and steam) and dipped in different concentrations of sulphites (SO₂), hydrogen peroxide (H₂O₂), ethylene‐di‐amine tetra‐acetic acid (EDTA) and citric acid for 10 min before smoking. Enzyme activities, colour characteristics, microbiological and sensory examinations were carried out every 2 weeks up to 8 weeks of storage in refrigerator at 4 °C. Results could be concluded that smoked mushroom pretreated with SO₂, H₂O₂ and steam blanching had the best colour values, the better score for all sensory characteristics and lower non‐enzymatic browning compared with other pretreatments. The most effective pretreatment against total aerobic bacteria and yeast & moulds were citric acid, EDTA and steam, and then smoking of mushroom can be attributed to the reduction of microbial counts. The most effective pretreatments on quality and safety of smoked mushrooms were H₂O₂ and steam. It can be concluded that thermal and chemical treatments, rather than smoking of mushroom, reduce enzyme activities and are suitable to preserve mushrooms.     

Effects of dietary rumen‐protected lysine on milk yield and composition in lactating cows fed diets containing double‐low rapeseed meal

This study investigated the effects of dietary rumen‐protected lysine (RPLys) on milk yield and composition in lactating cows fed diets containing double‐low rapeseed meal. Twenty‐four cows were divided into three groups and fed one of three dietary treatments: 10% soya bean meal (SBM) diet (control), 15% double‐low rapeseed meal (DLRSM) diet and 15% DLRSM + 32 g/d RPLys (DLRSML) diet. The results showed that no differences (P > 0.05) were observed in milk yield, 4% fat correction milk (FCM), energy correction milk (ECM), protein yield, milk fat yield, milk fat, lactose yield and lactose between control and DLRSM. Supplementation with DLRSML increased (P < 0.05) milk yield, 4% FCM, ECM, and protein yield compared with the SBM. The results indicated that DLRSM and DLRSML might be used to substitute for SBM as a protein source in lactating cows, and the latter might be more beneficial to improve the performance.     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

Effects of pretreatment ultrasound bath and ultrasonic probe,in osmotic dehydration,in the kinetics of oven drying and the physicochemical properties of beet snacks

Osmotic dehydration and ultrasound are pretreatments used in order to reduce costs and processing time in the drying of food. We investigated the effect of the ultrasonic bath and an ultrasonic probe in osmotic solution, as a pretreatment of the drying process in an oven, for beet snacks. Different conditions of pretreatments (TP: ultrasonic probe treatment; TB: ultrasonic bath treatment) were performed and analyzed for water loss (WL) and solid gain (SG). After the snacks were ready, we evaluated the drying kinetics, aw, Brix, color, texture, and anthocyanin content. No difference was observed between treatments for SG and WL. The pretreatments TP5, TP10 and TB20 decreased by 22.2% the drying time in the oven. Regarding the snacks, there was no difference in texture. The colors of TP5, TP10, and TB10 were similar to the control, as well as the a_w value of the TP5 and TP10. The TP5 had the highest anthocyanin content. The use of ultrasound probe in osmotic solution for 5 min, as a predrying treatment, is a viable technology as it reduces pretreatment and drying time without impairing the quality of the final product.

Practical applications

The use of ultrasound as a pretreatment in the drying of fruits reduces the drying time. Drying is an important process in obtaining new products, in addition to increasing shelf‐life.     

Combined ultrasound and heat pretreatment improve the enzymatic hydrolysis of clam (Aloididae aloidi) and the flavor of hydrolysates

The effects of pretreatments with ultrasound (UP), heat (HP), and combinations of heat-ultrasound (HP-UP) and ultrasound-heat (UP-HP) on the flesh enzymolysis of clam (Aloididae aloidi) and the flavor characteristics of hydrolysates were investigated. The optimum UP pretreatment conditions for the hydrolysis were obtained at ultrasound power of 500 W, ultrasound time of 20 min, and liquid/solid (L/S) ratio of 2:1 (v/w), yielded 41.33% of hydrolysis degree (DH). Among the different pretreatments, the UP pretreatment had the highest DH value, while the HP-UP pretreatment obtained the highest soluble peptide content. The UP pretreatment efficiently enhanced the contents of taste-active components, including free amino acids, 5′-nucleotides, and succinic acid. The HP pretreatment promoted the formation of pleasant volatile compounds and reduced the content of unpleasant volatile compounds, such as 1-octen-3-ol and 2-ethyl-1-hexanol. The combined HP-UP pretreatment was an effective method for improving the enzymatic hydrolysis of clam and the flavor of hydrolysate.     

逆流超声辅助酶解法制备大米ACE抑制肽

研究逆流超声预处理大米蛋白对其碱性蛋白酶酶解制备血管紧张素转换酶(Angiotensin-I Converting Enzyme,ACE)抑制肽的影响。首先从米渣中提取大米蛋白,以ACE抑制率为主要指标,水解度为辅助指标,运用单因素逐级优化法对酶解反应的底物浓度、时间、温度、加酶量和pH进行参数优化,在此基础上筛选逆流超声模式的最佳超声参数。结果表明最佳酶解参数为底物浓度30 g/L、加酶量(E/S)7.5%、温度50 ℃、pH8.5和酶解时间60 min,此时酶解产物ACE抑制率为45.59%,水解度为21.49%。最佳超声参数为超声频率20 kHz、功率密度170 W/L、时间12.5 min。此时酶解液ACE抑制率达72.24%,水解度为21.64%,相较于未超声组ACE抑制率提高了57.42%,相较于传统超声组,ACE抑制率提高了11.36%。结果表明逆流超声波辅助酶解法能有效提高酶解效率、减少能耗、促进ACE抑制肽制备。     

Determination of reaction kinetics of hydrolysis of tilapia (Oreochromis niloticus) protein for manipulating production of bioactive peptides with antioxidant activity,angiotensin‐I‐converting enzyme inhibitory activity and Ca‐binding properties

To manipulate enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein for production of bioactive peptides, its reaction kinetics was intensively studied. The study showed that the production of peptides with different bioactive properties including antioxidant activity, angiotensin‐I‐converting enzyme (ACE) inhibition and Ca‐binding property and their kinetics were affected by the degree of hydrolysis and substrate concentration. A comparative study on reaction kinetics found that the kinetic parameters for the production of each bioactive peptide are unique, that is, the maximum initial velocity, V_max, for hydrolysis of protein was as high as 1.07 mg mL^?1 min^?1, but that for the production of peptides with antioxidant activity and Ca‐binding property were very low, range of 7.14–66.7 μg mL^?1 min^?1, and that for the production of peptides with ACE inhibitory activity was the lowest, at 2.57 μg mL^?1 min^?1. This knowledge of reaction kinetics of protein hydrolysis would be useful for manipulating and optimising the production of peptides with desired bioactive properties.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

复合法提取高温菜籽粕中蛋白质的研究

以高温菜籽粕为原料,研究了淀粉酶法与碱提法结合提取蛋白质的工艺,其优化条件为:酶解pH7.0,酶解温度50℃,料液比1∶20,加酶量2.5%,酶解时间3 h;碱提pH10.0,碱提温度55℃,碱提时间2 h,料液比1∶12。在此条件下,菜籽蛋白质提取率达到71.63%。经等电点沉淀、70%乙醇脱色及冷冻干燥后,产品呈浅灰色,菜籽蛋白质得率为9.3%,单宁脱除率为56.65%,植酸脱除率为62.77%,硫甙脱除率达93.66%。     

酒糟纤维素超声波辅助酶解工艺研究

为促进我国酒糟资源的高值化开发和利用,本论文探究了基于超声波预处理的酒糟纤维素酶解工艺条件。首先通过单因素实验研究了超声工作参数（时间、温度、功率）及酶解工艺参数（时间、pH、温度、酶添加量、底物浓度）对酒糟纤维素酶解效果影响,在此基础之上进行了Plackett-Burman试验筛选出影响酶解反应的关键因素,再采用Box-Behnken试验对酶解工艺进行优化。Plackett-Burman试验结果表明,影响酒糟纤维素酶解的关键因素为:超声时间、酶解时间、酶解pH、酶添加量。Box-Behnken旋转中心组合试验优化得出最佳酶解工艺条件为:超声时间21 min、酶解时间140 min、酶解pH5.41、酶添加量990 U/g、超声温度60 ℃、超声功率200 W、酶解温度50 ℃、酶解底物浓度1:20 g/mL,在此条件下酒糟纤维素转化率为5.62%,较对照组（未经超声波处理）提高了35.4%,表明超声波预处理是提高酒糟纤维转化率的有效途径。     

Inhibition of lipase from rice (Oryza sativa) by diethyl-p-nitrophenyl phosphate

The inhibition of rice bran lipase (RBL) by diethyl-p-nitrophenyl phosphate was studied with reference to kinetics, nature of inhibition and also elucidate the effect of the inhibitor on the structure—function of the enzyme. Enzyme activity measurements shows that the inhibitor is more effective at 0.050 mM concentration of diethyl-p-nitrophenyl phosphate and the activity is 50% at this level of inhibitor concentration. The affinity of substrate for the enzyme was observed by the increase in the velocity of the reaction with increase in the substrate concentrations and double reciprocal plot indicates that the inhibition followed a competitive in nature and inhibition constant K _i is found to be 0.016 mM at pH 7.0. The decrease in apparent thermal denaturation temperature to 4 °C compared to control indicates the destabilization of enzyme in the presence of inhibitor. Fluorescence spectral measurements suggests that pronounced quenching of fluorescence intensity of RBL occurs at higher concentrations of diethyl-p-nitrophenyl phosphate and ‘K _a’ value was found to be 2.4 × 10⁴ M⁻¹ with free energy change ΔG^o—26 kJ/mol at 30 °C suggesting strong binding between the enzyme and the inhibitor with microenvironmental changes occur at the active site or in the neighbourhood of active site. The far UV-CD data suggest that there is no significant changes in the conformation of the enzyme as a result of binding of diethyl-p-nitrophenyl phosphate. These results indicate that diethyl-p-nitrophenyl phosphate is a inhibitor of RBL and binds to the enzyme in brining about inhibition without any structural alterations.     

PURIFICATION AND CHARACTERIZATION OF α‐GALACTOSIDASE FROM PEANUTS1

Alpha‐galactosidase was characterized in two peanuts market types, Runner and Spanish. The enzyme was purified 54 fold using ammonium sulfate precipitation, anion exchange chromatography and Size Exclusion High‐performance Liquid Chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated that the enzyme has a molecular weight of 30, 000 Da, and isoelectric focusing showed a pI of 5.2. The optimum temperature and pH were 50C and 6.0, respectively. The enzyme had a K_m of 0.221 mM when p‐nitrophenyl α‐D‐galactopyranoside (PNPG) was used as a substrate and 80.8 mM when raffmose was a substrate. Raffmose and galactose were found to be competitive inhibitors when PNPG was the substrate: K_i values were 25.4 and 189, respectively. The enzyme was very sensitive to Hg⁺⁺, Ag⁺⁺ and to a lesser extent to Cu⁺⁺. However, ethylcne diamine tetraacetic acid did not have an effect indicating no requirement for cations. The two peanut types tested showed identical enzyme activities.     

基于超声预处理的脱脂玉米胚芽ACE抑制肽的制备

为了研究超声辅助酶解制备血管紧张素转化酶(ACE)抑制肽的较优工艺,通过三种超声设备对脱脂玉米胚芽预处理,碱性蛋白酶酶解,酶解液体外模拟胃肠消化,以消化液ACE抑制率和酶解过程中玉米胚芽水解度(DH)为指标对超声预处理和酶解的参数进行单因素逐级优化。实验结果表明,最佳超声工作模式为20~40 kHz聚能式逆流双频交替超声模式;超声工作参数为功率密度120 W/L,超声预处理时间15 min,初始温度30℃,物料浓度5%;酶解条件为加酶量3000 U/g,酶解时间30 min,pH9.0,酶解温度50℃。在此条件下,酶解液的IC₅₀为4.166 mg/mL,比对照组降低了5.08%;胃肠消化液的IC₅₀为3.986 mg/mL,比对照降低了4.44%。制备的酶解产物,经模拟胃肠消化后具有较强的ACE抑制活性。优化获得的制备脱脂玉米胚芽ACE抑制肽的工艺是可行的。