Effect of ultrasonic-ionic liquid pretreatment on the hydrolysis degree and antigenicity of enzymatic hydrolysates from whey protein

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins.     

Effects of ultrasound synergized with microwave on structure and functional properties of transglutaminase-crosslinked whey protein isolate

In the present study, ultrasound (400 W, U), microwave heating (75 ℃ for 15 min, M) and ultrasound synergized with microwave heating (UM) pretreatments of whey protein isolate (WPI) were applied to investigate and compare their influence on structure, physicochemical and functional characteristic of transglutaminase (TGase)-induced WPI. From the results of size exclusion chromatography, it could be seen that all three physical pretreatments could promote the formation of polymers in TGase cross-linked WPI, whose polymer amounts were increased by the order of U, UM and M pretreatment. Among three physical methods, M pretreatment had the strongest effect on structure and functional characteristics of TGase-induced WPI. Furthermore, compared with TGase-induced WPI, α-helix and β-turn of M−treated TGase-induced WPI (M−WPI−TGase) were reduced by 7.86% and 2.93%, whereas its β-sheet and irregular curl were increased by 15.37% and 7.23%. Zeta potential, emulsion stability and foaming stability of M−WPI−TGase were increased by 7.8%, 59.27% and 28.95%, respectively. This experiment exhibited that M was a more effective pretreatment method than U, UM for WPI, which could promote its reaction with TGase and improve its functional properties.     

乳清分离蛋白-葡聚糖接枝物性质的荧光光谱法分析

乳清分离蛋白与葡聚糖的混合物在干热处理条件下,发生了以褐变为特征的美拉德反应。当葡聚糖分子量由67kD增至150kD时,游离氨基含量分别下降了35.77%和30.53%,糖链越长,其接入到蛋白质肽链的难度越大。采用荧光光谱对乳清分离蛋白-葡聚糖接枝物的性质进行分析。内源荧光光谱图显示,接枝产物在405nm的最大荧光强度显著提高,且350～500nm范围内的荧光强度顺序为:G67>G150,这说明接枝物中有Maillard反应体系所特有的荧光物质生成;由外援荧光光谱图得出,接枝产物在470nm的最大荧光强度均有明显降低,各溶液体系中荧光强度高低顺序依次为:WPI>G150>G67。疏水性指数的测定进一步说明两种不同分子量的葡聚糖接入到蛋白质肽链中,对乳清分离蛋白的疏水性均有一定的屏蔽作用。     

Effect of ultrasound treatment on interactions of whey protein isolate with rutin

Rutin is a biologically active polyphenol, but its poor water solubility and low bioavailability limit its application to the food industry. We investigated the effect of ultrasound treatment on the properties of rutin (R) and whey protein isolate (WPI) using spectral and physicochemical analysis. The results revealed that there was covalent interaction between whey protein isolate with rutin, and the binding degree of whey isolate protein with rutin increased with ultrasound treatment. Additionally, solubility and surface hydrophobicity of WPI-R complex improved with ultrasonic treatment, and a maximum solubility of 81.9 % at 300 W ultrasonic power. The ultrasound treatment caused the complex to develop a more ordered secondary structure, resulting in a three-dimensional network structure with small and uniform pore sizes. This research could provide a theoretical reference for studying protein–polyphenol interactions and their applications in food delivery systems.     

Thermosonicated whey protein concentrate blends on quality attributes of reduced fat Panela cheese

Aiming at producing a reduced fat cheese (RFC) as an alternative to full-fat Panela cheese, a highly consumed fresh Mexican dairy product, thermosonication (TS) processes (24 kHz, 400 W nominal power, 2, 4 and 6 min; 50, 55 and 60 °C) were evaluated to treat WPC (80% protein) blended with reduced-fat milk (1 and 2% fat), which were later LTLT pasteurized. TS blends were compared in terms of their technological properties (water holding capacity-WPC, gel firmness- GF, color, pH and titratable acidity) with those of a regular full fat (3%) LTLT pasteurized milk used as a control. Afterwards, a regression analysis was carried out with the obtained data in order to select the most appropriate conditions for cheesemaking purposes (similar GF, higher WHC with respect to the control), minimize both fat content and TS treatment duration to minimize energy expenses. According to these restrictions, the selected conditions were 1.5% fat milk-WPC blend, TS treated at 60 °C for 120 s; 1% fat milk-WPC blend, TS treated at 50 °C for 120 s and 1% fat milk-WPC blend, 50 °C for 144 s, which allowed preparing low fat cheeses (LFCs). These TS treatments were applied in a larger scale to elaborate Panela-type LFCs comparing different technological properties (cheese yield, syneresis, water content, texture profile analysis, color and titratable acidity) with those of a full fat variety, at day 1 and during 14 days of refrigerated storage. Results showed similar texture profiles of LFC cheeses and full fat milk cheeses throughout their storage period with significant changes in composition parameters (higher moisture, protein and salt contents, with low fat percentages), syneresis, selected color parameters (hue, b*), with no observed changes in cheese yield, TA and pH during cheese storage. These promising results are encouraging to develop LFCs with no physicochemical or technological defects using novel processing techniques that may help reducing calorie consumption without compromising sensory acceptability.     

High-intensity ultrasound pretreatment influence on whey protein isolate and its use on complex coacervation with kappa carrageenan: Evaluation of selected functional properties

The aim of this work was to evaluate the influence of high-intensity ultrasound (HIUS) treatment on whey protein isolate (WPI) molecular structure as a previous step for complex coacervation (CC) with kappa-carrageenan (KC) and its influence on CC functional properties. Protein suspension of WPI (1% w/w) was treated with an ultrasound probe (24 kHz, 2 and 4 min, at 50 and 100% amplitude), non HIUS pretreated WPI was used as a control. Coacervation was achieved by mixing WPI and KC dispersions (10 min). Time and amplitude of the sonication treatment had a direct effect on the molecular structure of the protein, FTIR-ATR analysis detected changes on pretreated WPI secondary structure (1600–1700 cm⁻¹) after sonication. CC electrostatic interactions were detected between WPI positive regions, KC sulfate group (1200–1260 cm⁻¹), and the anhydrous oxygen of the 3,6 anhydro-D-galactose (940–1066 cm⁻¹) with a partial negative charge. After ultrasound treatment, a progressive decrease in WPI particle size (nm) was detected. Rheology results showed pseudoplastic behavior for both, KC and CC, with a significant change on the viscosity level. Further, volume increment, stability, and expansion percentages of CC foams were improved using WPI sonicated. Besides, HIUS treatment had a positive effect on the emulsifying properties of the CC, increasing the time emulsion stability percentage. HIUS proved to be an efficient tool to improve functional properties in WPI-KC CC.     

油脂微胶囊壁材乳清蛋白与阿拉伯胶相互作用研究

利用傅里叶红外光谱法和计算机辅助分析法研究壁材乳清蛋白和阿拉伯胶在油脂微胶囊形成过程中的相互作用。结果表明,经高压均质和喷雾干燥后,乳清蛋白的酰胺A带向高波数方向移动,这可能是由于乳清蛋白和阿拉伯胶发生了共价交联,而酰胺Ⅰ带向高波数移动了6.1 cm-1则是由于蛋白质分子内的氢键作用力减弱所致。对酰胺Ⅰ带图谱进行高斯拟合后发现,乳清蛋白质二级结构中α-螺旋的含量由19.55%下降至17.50%,β-折叠的含量由30.59%下降至25.63%,共减少了7个百分点。这表明蛋白质分子内的氢键作用力减弱,致使蛋白质分子的刚性结构减弱,韧性结构增强,使蛋白质分子表现出一定的柔性。SDS-PAGE电泳研究结果表明,乳清蛋白-阿拉伯胶复合物中产生分子量较大的共价产物。喷雾干燥过程中乳清蛋白与阿拉伯胶发生了共价交联,使得复合物的乳化活性得到提高。用环境扫描电镜观测不同壁材制备的油脂微胶囊的表面结构,发现乳清蛋白-阿拉伯胶复合物为壁材制备的油脂微胶囊具有良好的韧性,微孔少,结构致密。     

Production of biologically active peptides by hydrolysis of whey protein isolates using hydrodynamic cavitation

Whey protein isolate (WPI) hydrolysates have higher solubility in aqueous phase and enhanced biological properties. Hydrolysis of WPI was optimized using operating pressure (ΔP, bar), number of passes (N), and WPI concentration (C, %) as deciding parameters in hydrodynamic cavitation treatment. The optimum conditions for generation of WPI hydrolysate with full factorial design were 8 bar, 28 passes, and 4.5% WPI concentration yielding 32.69 ± 1.22 mg/mL soluble proteins. WPI hydrolysate showed alterations in binding capacity over WPI. SDS-PAGE and particle size analysis confirmed the hydrolysis of WPI. Spectroscopic, thermal and crystallinity analyses showed typical properties of proteins with slight variations after hydrodynamic cavitation treatment. ABTS, DPPH and FRAP assays of WPI hydrolysate showed 7–66, 9–149, and 0.038–0.272 µmol/mL GAE at 1–10, 0.25–4, and 3–30 mg/mL concentration, respectively. Further, a considerable enhancement in fresh weight, chlorophyll, carotenoids, reducing sugars, total soluble sugars, soluble proteins content and total phenolics content was noticed during in vitro growth of sugarcane in WPI hydrolysate supplemented medium at 50–200 mg/L concentration over the control. The process cost (INR/kg) to hydrolyze WPI was also calculated.     

The effects of ultrasonic treated whey on the structure formation in food systems based on whey in combination with pectin and agar-agar

We studied the effects of ultrasonicated whey in food systems with the structure-forming additives such as pectin and agar-agar. The high-intensity (45KHz, 40 W with cavitation) ultrasonic treated whey was used. The conditions and optimal modes of cavitation based ultrasonic processing of curd milk whey have been determined. The mechanism of structure formation has been studied in detail.From the studies carried out, the scientific basis for the choice of structure-forming agents in food systems was established along with the range of rational concentrations of pectin and agar-agar. It was shown that in the case of processing milk curd whey by the cavitation method, the concentration of the structure former can be reduced by 2 times compared to using non-sonicated whey in the food system thus saving costs on the raw materials.It was established that high-purity cavity treatment minimizes gel-like food systems set time up to 20% compared to the control within 15 min. The duration of high-purity treatment within 15 min contributes to an increase in penetration pressure, which characterizes the texture of the gel-like food two times.     

FTIR分析共价偶联对抗体蛋白二级结构的影响

用共价偶联的方法制备了免疫胶乳,并利用傅里叶变换红外光谱技术,结合差谱、去卷积、二阶导和曲线拟合等计算机辅助分析方法,研究免疫胶乳中抗体蛋白的二级结构。结果表明,随着pH值的升高以及胶乳浓度的增大,抗体蛋白的有序结构含量增加。由此认为共价偶联会对抗体蛋白的二级结构产生较为显著的影响。     

Effect of ultrasound pretreatment on structural,physicochemical, rheological and gelation properties of transglutaminase cross-linked whey protein soluble aggregates

A solution (10%, w/v) of whey protein soluble aggregates (WPISA) was pretreated with high-intensity ultrasound (HUS, 20 kHz) for different durations (10–40 min) before incubation with transglutaminase (TGase) to investigate the effect of HUS on the structural, physicochemical, rheological, and gelation properties of TGase cross-linked WPISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that HUS increased the amounts of high-molecular-weight polymers/aggregates in WPISA after incubation with TGase. HUS significantly increased (P < 0.05) the degree of TGase-mediated cross-linking in WPISA, as demonstrated by a reduction in free amino group contents. HUS significantly increased (P < 0.05) the particle size, intrinsic fluorescence intensity, and surface hydrophobicity of TGase cross-linked WPISA, but had no significant impact (P > 0.05) on the zeta-potential or total free sulfhydryl group content of TGase cross-linked WPISA. The apparent viscosity and the consistency index of TGase cross-linked WPISA were significantly increased by HUS (P < 0.05), which indicated that HUS facilitated the formation of more high-molecular-weight polymers. HUS significantly increased (P < 0.05) the water holding capacity and gel strength of glucono-δ-lactone (GDL)-induced TGase cross-linked WPISA gels. The results indicated that HUS could be an efficient tool for modifying WPISA to improve its degree of TGase-mediated cross-linking, which would lead to improved rheological and gelation properties.     

FTIR analysis of protein secondary structure in cheddar cheese during ripening

Proteolysis is one of the most important biochemical reactions during cheese ripening. Studies on the secondary structure of proteins during ripening would be helpful for characterizing protein changes for assessing cheese quality. Fourier transform infrared spectroscopy (FTIR), with self-deconvolution, second derivative analysis and band curve-fitting, was used to characterize the secondary structure of proteins in Cheddar cheese during ripening. The spectra of the amide I region showed great similarity, while the relative contents of the secondary structures underwent a series of changes. As ripening progressed, the alpha-helix content decreased and the beta-sheet content increased. This structural shift was attributed to the strengthening of hydrogen bonds that resulted from hydrolysis of caseins. In summary, FTIR could provide the basis for rapid characterization of cheese that is undergoing ripening.     

FTIR investigation of the effects of ultra-strong static magnetic field on the secondary structures of protein in bacteria

Secondary structures of protein in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) exposed to the ultra-strong static magnetic field (SMF) were investigated by Fourier transformation infrared spectroscopy (FTIR). Difference index D value of amide I (1600–1700 cm⁻¹) showed that the ultra-strong magnetic field had little impact on S. aureus, but had strong impact on E. coli. The results indicated that 3.46–9.92% of the disorder coils in the secondary structures of protein in E. coli were turned into α-helices under SMF while applying deconvolution and curve fitting to amide I. At the same time, intermolecular β-sheets transforming into intramolecular ones suggested that cohesion among protein molecules had been destroyed and intramolecular hydrogen bonds strengthened. All the differences among the compositions of protein’s secondary structures in E. coli were mostly due to the varying degrees of various proteins affected by the magnetic field. The results may provide new insights into the structural changes of proteins induced by the SMF.     

The role of secondary structure in protein structure selection

The presence of highly regular secondary structure motifs in protein structure is a fascinating area of study. The secondary structures play important roles in protein structure and protein folding. We investigate the folding properties of protein by introducing the effect of secondary structure elements. We observed the emergence of several structures with both large average energy gap and high designability. The dynamic study indicates that these structures are more foldable than those without the effect of secondary structures.     

Ultrasound driven conformational and physicochemical changes of soy protein hydrolysates

The effect of ultrasound on the conformational and physicochemical properties of soy protein isolate hydrolysates (SPHs) was investigated. SPHs were prepared at hydrolysis times of 20 min, 60 min, and 180 min, then treated with ultrasound for 10 min, 20 min, and 30 min at a frequency of 20 kHz and output powers of 150 W and 450 W. The structural properties and antioxidant capacities of the aqueous layer of SPHs (ASPHs) after sonication were evaluated by Fourier-transform infrared spectroscopy (FTIR), intrinsic fluorescence, DPPH radical scavenging activity assays, and microscopy observations. Results obtained showed that ultrasound treatment significantly disrupted the peptide aggregates formed during protein hydrolysis. The protein solubility was significantly increased after sonication (by up to 18.33%), as did the percentage of proteins with MW < 1 kDa in ASPHs. The antioxidant capacity of ASPHs also increased, as measured by DPPH assay. FTIR analysis of ASPHs indicated that the protein secondary structures were different, with an increase in β-sheet and a decrease in α-helix and β-turn. Furthermore, the changes in fluorescence spectra of ASPHs showed the transition of protein tertiary structure with a greater exposure of Trp residues in the side chains. Scanning electron microscope (SEM) and atomic force microscope (AFM) observations of the morphological structure of ASPHs further confirmed the significant effect of sonication on disrupting peptide aggregates. In conclusion, ultrasound can be used as an efficient treatment to promote the solubility of protein hydrolysates.     

Functionalised dairy streams: Tailoring protein functionality using sonication and heating

Ultrasound can be used to modify the functional interactions between casein and whey proteins in dairy systems. This study reports on ongoing developments in understanding the effect of ultrasound and heating on milk proteins in systems with modified casein-whey protein ratios (97:3, 80:20 and 50:50), prepared from milk protein concentrates that were fractionated by microfiltration, based on protein size. Heating of concentrated casein streams (9% w/w) at 80.0 °C for up to 9 min resulted in reduced gelation functionality and increased viscosity, even in the absence of added whey proteins. 20 kHz ultrasonication at 20.8 W calorimetric power for 1 min was able to break protein aggregates formed during heating, resulting in improved gelation and reduced viscosity. Interestingly, when heated whey protein was recombined with unheated casein the gelation properties were similar to unheated controls. In contrast, when heat treated casein streams were recombined with unheated whey protein, the gel forming functionality was reduced. This study therefore shows that using specific combinations of heat and/or ultrasound, fractionated dairy streams can be tailored for specific functional outcomes.     

Effects of high hydrostatic pressure on secondary structure and emulsifying behavior of sweet potato protein

In this study, secondary structures of sweet potato protein (SPP) after high hydrostatic pressure (HHP) treatment (200–600?MPa) were evaluated and emulsifying properties of emulsions with HHP-treated SPP solutions in different pH values (3, 6, and 9) were investigated. Circular dichroism analysis confirmed the modification of the SPP secondary structure. Surface hydrophobicity increased at pH 3 and decreased at 6 and 9. Emulsifying activity index at pH 6 increased with an increase in pressure, whereas emulsifying stability index increased at pH 6 and 9. Oil droplet sizes decreased, while volume frequency distribution of the smaller droplets increased at pH 3 and 6 with the HHP treatment. Emulsion viscosity increased at pH 6 and 9 and pseudo-plastic flow behaviors were not altered for all emulsions produced with HHP-treated SPP. These results suggested that HHP could modify the SPP structure for better emulsifying properties, which could increase the use of SPP emulsion in the food industry.     

Influence of ultrasound-assisted ionic liquid pretreatments on the functional properties of soy protein hydrolysates

In this work, the effect of dual-frequency ultrasound-assisted ionic liquids (ILs) pretreatment on the functional properties of soy protein isolate (SPI) hydrolysates was investigated. The degree of hydrolysis (DH) of SPI pretreated by ultrasound and [BMIM][PF₆] increased by 12.53% as compared to control (P < 0.05). More peptides with low molecular weight were obtained, providing support for the changes in DH. The trichloroacetic acid-nitrogen soluble index presented an increase, suggesting a better protein hydrolysate property. The increase in the calcium-binding activity showed the ultrasound-assisted ILs pretreatment could potentially improve bone health. The foaming capacity and stability of SPI hydrolysates pretreated by ultrasound-assisted [BMIM][PF₆] always increased remarkably as compared to ultrasound-assisted [BDMIM][Cl] pretreatment. However, the synergistic effect of ultrasound-assisted [BMIM][PF₆] on the emulsifying activity and antioxidant activities (DPPH and hydroxyl radical scavenging activity) was not as ideal as ultrasound-assisted [BDMIM][Cl] pretreatment, which may be affected by the structure of peptide. In conclusion, these results indicated the combination of dual-frequency ultrasound and ionic liquids would be a promising method to improve the functional properties of SPI hydrolysates and broaden the application scope of compound modification in proteolysis industry.     

莲子蛋白组分二级结构的研究

对莲子蛋白质进行了Osborne蛋白质分类。采用傅里叶变换红外光谱(FTIR)对清蛋白、球蛋白、醇溶蛋白和谷蛋白进行二级结构分析。应用去卷积和曲线拟合方法对四种蛋白组分的酰胺Ⅰ和Ⅲ带进行分析,清蛋白和球蛋白之间以及醇溶蛋白和谷蛋白之间各相应子峰峰位和二级结构峰面积百分比差异较小,但前两者各相应子峰峰位与后两者略有差异;而前两者各相应二级结构峰面积百分比与后两者有较大差异,特别是前两者的各相应有序结构(α-螺旋+β-折叠)峰面积的百分比明显大于后两者。用0.1 mol.L-1NaCl溶液提取的球蛋白和清蛋白有序结构含量均在55%左右,而醇或碱提的醇溶蛋白和谷蛋白的有序结构含量仅为40%左右,盐提的蛋白质二级结构有序性和稳定性更高。     

Effect of ultrasonic power on properties of edible composite films based on rice protein hydrolysates and chitosan

Rice protein hydrolysates (RPH) are incapable of film formation by self-crosslinking due to low molecular mass. Hence, we used chitosan (CS) as a modifier and developed rice protein hydrolysates/chitosan (RPH/CS) edible composite films by means of ultrasound. Results showed that ultrasound treatment decreased the particle size and the viscosity of film-forming solutions. The value of elongation at break of composite films was increased by 125% at 400 W compared with untreated film. The peroxide value of soybean oil was significantly reduced from 16.99 ± 0.78 meq/kg to 2.23 ± 0.09 meq/kg with the increase of ultrasonic power. Ultrasound treatment was efficient in keeping smooth on surface, and the films at ultrasound treatment of 200 W had better compatibility. Moreover, hydrogen bonds and covalent interactions were probably the main forces between RPH and CS and contributed to film formation under ultrasound treatment, which supported by analyses of Fourier transform infrared spectroscopy and X-ray diffraction. These results suggested that ultrasound was an effective method to improve the properties of edible composite films.