Facile optical resolution of amino acid esters via hydrolysis by an industrial enzyme in organic solvents

Racemic amino acid esters were optically resolved via hydrolysis in organic solvents by the catalysis of an industrial alkaline protease, “Alcalase”. The products which were composed mainly of L-amino acids were insoluble and easily separated by filtration. The activity of the enzyme and enantiomeric excess of the products were significantly dependent on the nature of solvent and the water content in the reaction media. Generally, high values of enantiomeric excess were obtained at low water contents. Many natural and unnatural amino acids were resolved by this method.     

Bioaccessible Iron and Zinc in Native and Fortified Enzyme Hydrolyzed Casein and Soya Protein Matrices

Casein and soybean are superior quality proteins; however, these are known to inhibit mineral absorption. These investigations were aimed at enzymatic modification of these proteins and to study their effect on mineral bioaccessibility. Casein and soybean proteins were hydrolyzed with alcalase and trypsin individually under optimum conditions. The protein hydrolysates, prepared with different degrees of hydrolysis, were freeze dried, fortified with either iron or zinc and analyzed for bioaccessible minerals in vitro. Proteolytic hydrolysis enhanced the bioaccessibility of iron and zinc in proportion to the degree of hydrolysis. Tryptic hydrolysis enhanced the bioaccessible iron from 1–4% in casein and from 1.3–3.3% in soybean. Alcalase hydrolysis showed a comparatively higher enhancement with both the proteins. Tryptic hydrolysis enhanced zinc bioaccessibity by 3-fold in casein and alcalase hydrolysis enhanced by 2- to 2.5-fold. In soybean, dephytinization showed a synergistic effect. In conclusion, enzymatic hydrolysis of proteins looks promising for enhancing bioaccessibility of minerals in protein matrices.     

黄粉虫幼虫粉酶解及体外抗氧化性研究

主要研究了Alcalase蛋白酶水解脱脂黄粉虫幼虫粉的工艺,并对水解产物的体外抗氧化能力进行测定.在单因素基础上以还原力为指标设计正交试验得到水解最佳条件为:温度60℃,pH值8.5,加酶量150μL,反应时间100 min.抗氧化试验结果表明,脱脂黄粉虫幼虫粉水解液清除1,1-二苯基-2-苦基苯肼(DPPH)自由基和羟自由基的半抑制率(IC_(50))分别为0.45 mg/mL和4.1 mg/mL,分别是VC的IC_(50)值的112.5倍和19.5倍,还原能力是VC的1/100.将水解前后的脱脂黄粉虫幼虫粉溶液进行高效毛细管电泳(HPCE)测定,测定结果表明,经Alcalase蛋白酶水解后,溶液中可溶性组分增多.     

Alcalase水解酪蛋白制备磷酸肽和非磷肽的研究

研究了Alcalase水解酪蛋白制备磷酸肽和非磷肽的作用条件,同时分析了酪蛋白磷酸肽(CPPs)和酪蛋白非磷肽(CNPPs)的相对分子质量分布和氨基酸组成。结果表明,Alcalase水解酪蛋白的最佳条件是:底物浓度5%,加酶量0.03mL/g蛋白质,温度60℃,pH9.0。乙醇浓度和酪蛋白的水解度(DH)对CPPs的得率和N/P(摩尔比)均有影响:当乙醇浓度为70%,DH为18%时,CPPs得率达到最高,为23.94%,N/P(摩尔比)是9.61,CNPPs得率为76.06%。CPPs和CNPPs的相对分子质量都分布在200～5000之间,其中低于700的肽含量最高,分别为61.52%和67.26%。CNPPs中疏水性氨基酸含量远高于CPPs。     

Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Soybean protein hydrolysates were prepared by hydrolyzing soybean protein isolates with a protease alcalase to a degree of hydrolysis of 16.6%, and then modified by alcalase-catalyzed plastein reaction to reveal the impact of plastein reaction on the ACE-inhibitory activity of the modified product in vitro. The suitable conditions of plastein reaction of soybean protein hydrolysates were selected based on the results of response surface methodology with the decreased amount of the free amino groups of the modified product as response. When reaction temperature was fixed at 30°C, the selected conditions were as follows: concentration of soybean protein hydrolysates of 45% (w/w), addition level of alcalase of 275 U/g peptides, and reaction time of 3 to 4 h. Soybean protein hydrolysates and eight modified products were evaluated for their ACE-inhibitory activities in vitro. The assay results highlighted that plastein reaction improved the ACE-inhibitory activity of the modified product. The IC₅₀ of the modified products ranged from 0.64 to 1.11 mg/mL, while that of soybean protein hydrolysates was 1.45 mg/mL. The decreased amount of the free amino groups of the modified product showed influence on the ACE-inhibitory activity in vitro. Analysis results from size exclusion chromatography confirmed that some plasteins with higher molecular weights were formed in the modified product. Our results showed that alcalase-catalyzed plastein reaction could be applied as a potential approach to enhance the ACE-inhibitory activity of soybean protein hydrolysates in vitro.     

大米浓缩蛋白酶法改性与功能性质评价

以大米浓缩蛋白（rice protein concentrate,RPC）为原料,利用碱性蛋白酶（Alcalase）改性。探讨了在最适条件下,即底物浓度5%、酶与底物比例0.8%、pH8.5、55℃,经过改性制得改性大米浓缩蛋白（modified rice protein concentrate,MRPC）的功能性质：氮溶性指数（NSI）为52.9%、乳化活性（EA=0.101）、乳化稳定性（ES=26.7%）、表面疏水性（2627.6）以及分子量分布。研究表明,经过Alcalase改性后的MRPC与大豆分离蛋白接近,具有作为食品蛋白质添加剂开发的潜力。     

Immunomodulatory properties of the milk whey products obtained by enzymatic and microbial hydrolysis

The objective of this study was to investigate the in vitro immunomodulatory effect of milk whey through enzymatic hydrolysis, microbial fermentation and two‐stage hydrolytic reactions (enzymatic and microbial reactions) by analysing cytokine profiles. Results indicated that the milk whey sample fermented by Lactobacillus kefiranofaciens M1 and two‐stage hydrolytic reactions (hydrolysed by Alcalase and then fermented by L. kefiranofaciens M1) could significantly induce the production of tumour necrosis factor (TNF)‐α and IL‐12 compared with the milk whey control and enzymatic treatments. Further characterisation of the immunomodulatory factor by membrane filtration and mutanolysin hydrolysation, the stimulatory activity for IL‐12 and TNF‐α production was found to be reduced and to be correlated positively with the cell wall components in L. kefiranofaciens M1. In addition, Th2‐polarised splenocytes revealed that L. kefiranofaciens M1 had both IL‐12 inducing and IL‐4 repressing activities. These results suggested that L. kefiranofaciens M1 could direct the Th1/Th2 balance toward Th1.     

The effect of enzyme systems and processing on the hydrolysis of peanut (Arachis hypogaea L.) protein

In vitro protein digestion studies were carried out on raw and roasted peanut flour as the starting material in the production of peanut protein hydrolysate. Peanut flour was hydrolyzed with alcalase and alternately in a sequential digestion with pepsin-pancreatin, both for up to 24 h. The degree of hydrolysis (DH) at different times of hydrolysis was determined using the trinitrobenzenesulfonic acid (TNBS) method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to indicate destruction of native protein units in the enzymatic digests.Hydrolysis with alcalase was very rapid for the first 6 h after which a plateau was reached, whereas that with pepsin–pancreatin was more gradual reaching a plateau after 12 h of hydrolysis. Raw peanut hydrolyzed with alcalase and pepsin–pancreatin had 23% and 21% DH after 24 h respectively, whilst roasted peanut hydrolyzed with alcalase had 21% DH, with the pepsin–pancreatin hydrolysate recording the highest value of 25% after 24 h of hydrolysis.SDS-PAGE results showed that raw peanut samples behaved differently from the roasted samples; increasing hydrolysis time reduced larger peanut protein subunits, with only peptides of <20 kDa visible after hydrolysis for raw peanut, and virtually no distinct visible bands for the roasted peanut after 3 h of hydrolysis.     

Alcalase蛋白酶降解大豆胰蛋白酶抑制剂的研究

研究了不同酶解条件下 (pH值、温度、时间、加酶量和添加巯基还原剂 ) ,碱性内切蛋白酶Alcalase对大豆蛋白和大豆胰蛋白酶抑制剂的降解作用。研究结果表明 ,Alcalase可同时降解大豆蛋白和胰蛋白酶抑制剂。该酶解反应的最适条件为 :pH 8 0、温度 6 0℃、最适加酶量 10 μL/g蛋白 (约 0 0 2 832AU/ g蛋白 ) ,添加Na2 SO3为ω(Na2 SO3) =0 3% ,水解时间 4h。在此条件下 ,残留胰蛋白酶抑制活性为对照的 2 0 % ,可溶性蛋白含量可达 2 7mg/mL ,游离氨基酸含量为 7 1mg/mL ,大豆蛋白的水解度为 8 9%。还讨论了Alcalase蛋白酶降解大豆蛋白生成小肽的最佳反应条件     

醇法大豆浓缩蛋白酶法改性研究

为提高醇法大豆浓缩蛋白的溶解性，采用Alcalase蛋白酶对醇法大豆浓缩蛋白进行酶法改性试验。试验表明，酶法水解能显著提高大豆浓缩蛋白的溶解性。酶解的最佳条件是pH8．5、温度62℃、底物浓度5％，酶浓度2％(E／S)，在此条件下酶解4h，大豆浓缩蛋白的水解度在12％以上，大豆浓缩蛋白的NSI从10％提高到85％左右，有较好的溶解性。并利用浊度法测定了不同水解度条件下酶解大豆浓缩蛋白的乳化特性，结果表明水解度约为8％时乳化性最大，水解度约为6％时乳化稳定性最好。