Succinylated whey protein isolate as a sustained-release excipient of puerarin derivative oral tablets:Preparation,optimization and pharmacokinetics

This work was done to investigate succinylated commercial whey protein isolate(S-WPI)as an oral sustained-release delivery carrier for puerarin 5(PR-5). The succinylation condi-tions were established for S-WPIs by optimization of single factor study and Box–Beehnkendesign. The effect of succinylation degree on S-WPIs solubility was evaluated. Physicochem-ical properties of S-WPIs dried by different three methods on their flow ability, particle size,morphology and in vitro release behavior were studied. After preparing PR-5 sustained re-lease protein tablets with S-WPIs as the carrier by direct powder compression method, thedrug release were studied in vitro and the oral pharmacokinetics and bioavailability wasevaluated using in vivo dog model. It was observed that concentration of substrate has asignificant effect on succinylation. Release behavior in vitro showed spry dried S-WPIs with100% succinylation rate and 30% drug loading would be applied to the preparation of PR-5 sustained-release protein tablets based on the swelling mechanism(protein loss). Comparedwith PR-5 conventional tablet with oral administration, T max value of PR-5 sustained-releaseprotein tablets was approximately 1.58 fold greater than those of the conventional tablets asfurther evidenced by the significantly prolonged MRT and T 1/2. The findings demonstratedthat spray-dried S-WPIs has potential as a promising functional excipient for the design of PR-5 oral sustained-release tablets which can fully improve sustained-release effect and oral bioavailability.

Influence of particle size and ionic strength on the freeze-thaw stability of emulsions stabilized by whey protein isolate

The influence of particle size and ionic strength on the freeze-thaw(FT) stability of emulsions stabilized by whey protein isolate(WPI) was investigated in this study. The destabilization of emulsions during the FT process could be suppressed in a way by decreasing the particle size of the initial emulsions, which was the result of retarding the coalescence between oil droplets. To further improve the FT stability of emulsions, different amounts of Na Cl were added before emulsification. The emulsions with the ionic strength at 30–50 mmol/L exhibited good FT stability. Notably, the ionic strength in this range would not lower the freezing point of emulsions below the freezing temperature used in this study. Salt addition could improve the structural properties of proteins, which was available to strengthen the rigidity and thickness of interfacial layers, sequentially building up the resistance that the destruction of ice crystals to emulsions. Moreover, stronger flocculation between emulsion droplets could promote the formation of a gel-like network structure dominated by elasticity in the emulsion system, which might effectively inhibit the movement of droplets, and improve the FT stability of emulsions eventually. The result was of great significance for the preparation of emulsion-based foods with improved FT stability.

糖基化对分离乳清蛋白结构和功能特性的影响

为确定糖基化处理分离乳清蛋白的最佳工艺,研究其结构和功能特性,以分离乳清蛋白(WPI)与沙蒿胶(ASG)为原料,进行糖基化反应。通过响应面法确定改性WPI最佳工艺参数为:时间118 min,温度90℃,反应初始pH 12,在该条件下乳化活性为0.735,乳化稳定性为245.103。在最佳工艺参数条件下,制备糖基化产物,结果表明,糖基化产物(WPI-ASG)与单一蛋白相比,微观结构差异显著,乳液粒径分布相似,参与反应的主要游离氨基酸赖氨酸(Lys)和精氨酸(Arg)分别减少了23%和31.1%,荧光强度降低,FTIR-ART光谱图显示对应吸收峰有明显的减弱或增强,SDS-PAGE测得泳道强度下降,差示扫描热量仪测得峰值温度和热焓值升高。综合分析表明,WPI-ASG具有更好的乳化性和热稳定性。本研究结果对分离乳清蛋白的乳化性能提升,蛋白-多糖共价复合物的制备,物理稳定性的提高,应用蛋白或糖类的开发,具有一定参考意义。

Gastrointestinal digestive fate of whey protein isolate coated liposomes loading astaxanthin:Lipolysis,release,and bioaccessibility

Whey protein isolate coated astaxanthin-loaded liposomes were prepared in this work.The gastrointestinal digestive fate of whey protein isolate coated astaxanthin-loaded liposomes was evaluated in terms of particle size,size distribution,zeta potential,and morphology during in vitro digestion as a function of time.Analysis on the particle size and morphology of whey protein isolate coated astaxanthin-loaded liposomes showed that the majority of particles maintained spherical shape with a progressive increase of particle size after passage through the digestion.The zeta potential on whey protein isolate coated astaxanthin-loaded liposomes became highly negative after digestion.As compared in uncoated liposomes,the astaxanthin release in whey protein isolate coated liposomes was slower in simulated gastric fluid digestion,while was faster in simulated intestinal fluid digestion.Through in vitro digestion,the bioaccessibility of astaxanthin was improved significantly after whey protein isolate coating.It was also found that the whey protein isolate coating could protect liposomes against destruction and suppress the lateral mobility of pyrene,resulting in lower micropolarity and fluidity of liposomal membrane during the digestion.These findings may guide the potential application of whey protein isolate coated liposomes for improving the bioaccessibility and stability of astaxanthin in nutraceuticals and pharmaceutics.

Combination of microwave heating and transglutaminase cross-linking enhances the stability of limonene emulsion carried by whey protein isolate

In this study,we examined the effect of transglutaminase(TG)cross-linking time(0 h,2 h,4 h,8 h,and 12 h)on the stability of limonene emulsion loaded by microwave-heated WPI(MWPI).Size exclusion chromatography results showed that molecular weight of TG-treated MWPI and WPI was enhanced with the increase in TG cross-linking time(0–12 h).Microwave and TG cross-linking decreased particle size(7.55%)and increasedζ-potential(5.33%)and protein adsorption(4.58%)of WPI emulsion at TG cross-linking time of 12 h.CLSM results showed smaller droplet size of MWPI-TG emulsion and its uniform size.The obtained limonene emulsion loaded by MWPI-TG at 12 h showed the lowest creaming index and the strongest oxidative stability under aerobic conditions after 10th days of storage.Thus,the combination of microwave heating and TG cross-linking effectively enhanced the stability of limonene emulsion stabilized by WPI and facilitating the application of WPI in the fields of food,medicine,cosmetics,and other areas.

Physicochemical properties and antibacterial application of silver nanoparticles stabilized by whey protein isolate

Nano metal materials have been widely explored to be applied in medical,environmental,and material science.Among these nanoparticles,especially silver nanoparticles(AgNPs),have drawn increasing attention for antimicrobial applications.Most researchers are keen on the development of the biologically friendly capping reagents for the synthesis of AgNPs,instead of unfriendly organic polymers.In this study,the liquid chemical reduction method was used to synthesize AgNPs with edible whey protein isolate(WPI)as a capping reagent.These WPI-AgNPs had a broad size distribution(average diameter of 138.6 nm),and their dimensions could be readily controlled in the range of 22.5-149.6 nm by introducing different concentrations of chloride.Subsequently,it was confirmed that WPI-AgNPs were formed through two mechanisms,which were respectively reduced in situ(without the addition of NaCl)and ex situ(in presence of NaCl)to yield silver nanoparticles.The WPI-AgNPs synthesized in presence of 10 mM of NaCl as mediation reagent were stable at room temperature or 4℃ up to 3 months.Furthermore,the synthesized WPI-AgNPs had a good antibacterial activity toward pathogens including Gram-negative E.coli and Gram-positive S.aureus.The results shed light on method and capping reagent to stabilize silver nanoparticles,which highlighted the potential of WPI and chloride in metal nanoparticle synthesis.

美拉德反应对乳清分离蛋白及其水解物抗氧化性的影响

以乳清分离蛋白及其蛋白水解物为原料分别与半乳糖发生美拉德反应,研究两者美拉德反应的褐变程度、接枝度及产物荧光光谱的变化,同时以乳清分离蛋白和蛋白水解物作对照,研究两者美拉德反应产物的抗氧化性。结果表明:乳清分离蛋白及其水解物美拉德反应的褐变程度、接枝度及产物的抗氧化性均在4 h达到最大。其中,乳清分离蛋白及其水解物与半乳糖美拉德反应的褐变程度和接枝度最大值分别为1.114、18.431%和1.413、28.273%;两者美拉德反应产物的还原力、2,2’-联氨-二(3-乙基-苯并噻唑-6-磺酸)二铵盐自由基清除能力和1,1-二苯基-2-三硝基苯肼自由基清除能力分别为0.605、46.29%、61.77%和0.923、69.81%、78.43%,均显著高于对照组(P<0.05)。同时,内源荧光光谱发现,美拉德反应改变了乳清分离蛋白及其水解物的构象,使二者的结构更加松散。

抗氧化剂对羟自由基引起的乳清分离蛋白氧化抑制效果的研究

采用羟自由基对乳清分离蛋白(WPI)进行氧化,并采用在氧化前添加抗氧化剂的方法,研究其对乳清分离蛋白氧化的抑制作用。实验分为3组,即不添加抗氧化剂的对照组、添加α-生育酚和丁羟基茴香醚(BHA)的处理组,测定氧化1、5h和12h后WPI羰基、总巯基、二聚酪氨酸、表面疏水性和SDS-PAGE的变化。结果表明,蛋白氧化会显著提高WPI中的羰基含量和二聚酪氨酸含量(P<0.05),而且会使蛋白质的疏水性增加(P<0.05)。添加α-生育酚和BHA的乳清蛋白氧化5h,与对照组相比可以使羰基含量降低35.11%和50.15%,巯基含量增加27.73%和38.82%。通过SDS-PAGE表明,添加抗氧化剂可以减少蛋白质的聚合。抗氧化剂的添加抑制了乳清蛋白的氧化。

蛋白质-多糖复合物对β-胡萝卜素乳液的影响

研究高温条件下,不同反应时间形成的乳清分离蛋白(WPI)-甜菜果胶共价复合物对β-胡萝卜素乳液稳定性的影响。研究发现,WPI与甜菜果胶在80℃,反应5 h后形成的β-胡萝卜素乳液粒径较小,稳定性较好,其在高温、冷冻-解冻、高离子强度、低酸各种不同加工条件下明显改善β-胡萝卜素乳液稳定性。

乳清分离蛋白与壳聚糖美拉德反应初级阶段产物乳化性研究

采用干热美拉德反应制备乳清分离蛋白(WPI)-壳聚糖复合物,通过对复合物的乳液粒径及稳定性分析,研究反应温度、WPI与壳聚糖质量比及反应时间对复合物乳化性的影响。荧光光谱分析表明:WPI和壳聚糖在干热反应后发生共价交联并且结构发生了变化。WPI与壳聚糖在50℃、相对湿度79%干热条件下,反应1～4d的产物为Maillard反应初级阶段产物,该产物的乳化性质得到改善。其中,WPI与壳聚糖以质量比1:4混合,50℃反应1d的产物乳化稳定性是反应前的10倍。

微波加热对牛乳清蛋白氧化的影响

本研究旨在探究普通加热和微波加热对牛乳清分离蛋白(WPI)氧化影响的差别。分别比较普通方式加热15、30、60和90min以及微波加热60、120、240和360s后WPI的羰基含量、巯基含量、二聚酪氨酸水平、表面疏水性、荧光光谱和SDS-PAGE电泳的变化。结果表明,与普通加热相比,较长时间(>120s)的微波加热会使WPI的羰基含量、二聚酪氨酸含量明显的增加,巯基含量明显下降,表面疏水性与荧光光谱结果显示长时间微波加热可能对WPI结构有明显修饰作用并改变其空间构象,SDS-PAGE结果显示长时间微波加热有明显的交联现象产生。

自由基氧化引起乳清蛋白溶解性、凝胶强度和乳化性变化的研究

主要研究了在羟基自由基氧化体系中,不同H2O2浓度(1～20mmol/L)及不同FeCl3浓度(0.1～2mmol/L)对乳清蛋白溶解性、凝胶强度和乳化性等功能特性的影响。每种氧化条件的氧化时间分别为1、3、5h。结果表明:氧化对蛋白功能性起着很大的破坏作用,同未氧化的对照组乳清蛋白相比,氧化后乳清蛋白在所有氧化条件下,溶解性降低超过35%、凝胶强度降低达85%以上,在不同的氧化剂浓度下,5h氧化后乳清蛋白的乳化性均降低达60%以上,FeCl3浓度的改变对功能性的破坏更大。并且发现,溶解性的降低直接影响着蛋白的凝胶性和乳化性。总之,氧化会破坏蛋白质的结构,降低乳清蛋白的功能特性,但是适当的氧化可以提高乳化性。在实际生产中应该合理控制蛋白氧化的发生。

不同微波预处理对苹果汁褐变特性的影响

为了抑制苹果汁在加工过程中的褐变,将微波加热技术与乳清分离蛋白(WPI)、抗坏血酸(AA)、柠檬酸(CA)三种褐变抑制剂相结合,采用不同的微波功率和时间处理苹果原料,研究不同预处理方式对苹果汁褐变特性的影响。结果表明,随着微波功率提高和处理时间的延长,苹果汁多酚氧化酶(PPO)活性降低,色泽提高;当功率为720W,处理时间120s时,果汁PPO几乎被完全抑制,色泽、总酚、氨基态氮含量增加,总糖含量下降,总酸含量略微提高。其中,经WPI水浴微波处理后的果汁L值为91.14,与其他预处理后的果汁色泽相差不大,且总酚含量增幅最小,因而是较佳的抑制果汁褐变方法。

自由基氧化引起乳清蛋白理化性质变化的研究

研究了在羟基自由基氧化体系中,不同H2O2浓度(1～20 mmol/L)及不同FeCl3浓度(0.1～2 mmol/L)对乳清蛋白羰基、巯基、二聚酪氨酸等理化性质的影响。每种氧化条件的氧化时间分别为1,3,5 h.。结果表明:氧化显著地影响了乳清蛋白的理化性质,同未氧化的对照组乳清蛋白相比,经过5 h氧化,所有浓度的FeCl3体系中,羰基增加3倍以上;所有条件下的巯基损失均达40%以上;在浓度为20 mmol/L的H2O2或2 mmol/L的FeCl3中,二聚酪氨酸分别增加了5倍和7倍。并且发现,在不同的FeCl3条件下其变化趋势更为迅速。由此可知,氧化极大程度地改变了蛋白的理化性质,并可能导致蛋白结构的改变,进而可能影响其功能性质。

羟基自由基氧化体系对乳清蛋白、β-乳球蛋白化学结构的影响

本实验主要研究乳清蛋白(WPI)和β-乳球蛋白(β-Lg)经过FeCl3/抗坏血酸(AsA)/H2O2产生的羟基自由基氧化系统氧化后化学结构产生的变化。两种蛋白分别经过0.1mmol/L或者1mmol/LFeCl3氧化1、5和12h后,总巯基、游离氨都下降,而羰基、二聚酪氨酸和疏水性都呈增加的趋势。低Fe3+浓度氧化1h,WPI巯基含量降低38.5%,β-Lg降低11.6%;而游离氨分别降低20.68%和0.64%。高Fe3+浓度氧化5h,WPI羰基增加32.4%,β-Lg增加8.4%;二聚酪氨酸分别增加132.4%和28%;疏水值增加161.1%和0.7%。高Fe3+浓度带来的氧化效果要比低Fe3+浓度明显(p<0.05)。这说明,氧化改变了蛋白的化学结构,氧化程度取决于浓度Fe3+的浓度,且β-乳球蛋白比乳清蛋白有更好的稳定性。

低酯果胶和乳清分离蛋白复合凝胶性质的研究

本实验研究混合体系pH值、NaCl浓度和CaCl2浓度对低酯果胶与乳清蛋白复合凝胶的硬度和持水能力的影响。响应面分析结果表明,pH值、NaCl浓度和CaCl2浓度对凝胶性质有显著影响。低酯果胶和乳清分离蛋白复合凝胶的最佳条件为pH6、NaCl浓度0.2mol/L、CaCl2浓度10mmol/L,在此条件下制得的复合凝胶强度接近200g,持水能力接近75%。

羟基自由基氧化对乳清蛋白氨基酸含量的影响

主要研究了在羟基自由基氧化体系中,不同的H2O2浓度(1～20mmol/L)和FeCl3浓度(0.1～2mmol/L)下对乳清蛋白羰基、氨基和氨基酸含量的影响,每种氧化条件的氧化时间分别为1、3、5h。结果表明:氧化导致了蛋白中羰基含量的增加和氨基含量的降低,并且显著地影响了乳清蛋白的氨基酸含量。同未氧化的对照组乳清蛋白相比,总氨基酸和必需氨基酸含量均发生了明显降低,并且随着氧化时间的增加降低越迅速。总氨基酸含量最多降低达13%,必需氨基酸降低达16.2%,尤其在FeCl3体系表现更为显著。同时也发现,氨基酸的变化与羰量和氨基的变化是密切相关的。因此,在实际生产中我们应尽量控制蛋白氧化的发生,以减少氨基酸等营养成分的损失。

乳清蛋白抗氧化肽对H_2O_2所致人胚肺成纤维细胞MRC-5过氧化损伤的保护作用

【目的】探讨纯化后的乳清蛋白抗氧化肽P4对人胚肺成纤维细胞(human lung fibroblast)MRC-5过氧化损伤的保护作用及可能的作用机制。【方法】采用H2O2诱导建立细胞氧化损伤模型,应用四唑蓝快速比色法(MTT法)检测细胞存活率,通过检测细胞培养液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量,来确定P4对过氧化损伤MRC-5细胞的保护作用,并利用电镜观察细胞形态学变化。【结果】1mmol·L-1H2O2孵育24h可显著诱导MRC-5细胞损伤,使细胞存活力下降到22.47%,细胞经不同浓度的抗氧化肽P4(4、20、100μg·mL-1)与H2O2共孵育后,特别是100μg·mL-1(高剂量组)抗氧化肽P4可使细胞存活率达到44.77%。同时,提高乳清蛋白抗氧化肽P4的浓度,可促进受损的MRC-5细胞修复,提高了SOD、CAT、GSH-Px酶活性,降低MDA含量。扫描电镜和透射电镜观察结果也表明,一定浓度的乳清抗氧化肽对MRC-5细胞具有保护作用。【结论】乳清抗氧化肽通过拮抗H2O2而对MRC-5的过氧化损伤具有保护作用。

自由基氧化的乳清蛋白在冰淇淋中的应用

研究经羟基自由基产生体系氧化后的乳清蛋白在冰淇淋中的应用,测定蛋白氧化的主要指标羰基含量,并对影响冰淇淋品质的主要参数(包括膨胀率、抗融性、保形性)以及感官指标进行评定。结果表明:在冰淇淋中添加经H2O2体系氧化后的乳清蛋白,产品各项指标明显好于添加FeCl3体系的冰淇淋,且能够提高产品的性能,尤其是添加氧化1h的氧化蛋白其感官评分达到最大,但同对照比口感略有降低。这说明,适当的氧化可以改进产品的品质,提高产品的可接受性,但需改进口感。