分步酶解小麦面筋蛋白制备低苦味肽粉的研究

目的 研究分步酶解小麦面筋蛋白(wheat gluten, WG)制备低苦味肽粉的工艺。方法 选用中性蛋白酶、木瓜蛋白酶、胃蛋白酶水解WG至8%水解度,接着用风味蛋白酶对水解产物进行脱苦处理,对不同酶解产物中苦味肽的特性进行系统研究,探究苦味肽含量、氨基酸组成、分子量分布、表面疏水性等指标变化对WG酶解物苦味值的影响,对比风味蛋白酶对不同单酶酶解物的脱苦效果差异,分析风味蛋白酶对WG酶解物脱苦的内在机理,进而确定制备低苦味小麦蛋白肽粉的最佳酶解工艺。结果 中性蛋白酶的酶解产物经风味蛋白酶作用后,脱苦效果最显著,苦味肽苦味值从4.08降至2.25,酶解产物的苦味值可下降56.42%。木瓜蛋白酶的酶解产物经风味蛋白酶作用4 h后,酶解产物的苦味值最低,制备出苦味值为1.28的WG低苦味肽粉。结论 经分步酶解作用后,酶解产物中苦味肽的含量下降;疏水性氨基酸比例的下降和游离氨基酸含量的升高引起苦味肽苦味阈值的增大,共同导致酶解产物苦味值显著降低,该研究为酶解脱苦技术的快速发展和WG活性肽工业化生产提供新的参考。     

Identification of an Angiotensin I-converting Enzyme Inhibitory Peptides from Protein Hydrolysates by a Soybean Protease and the Antihypertensive Effects of Hydrolysates in 4 Spontaneously Hypertensive Model Rats

ABSTRACT: Our previous study demonstrated that, because of its substrate specificity, protein hydrolysates by protease D3, which is originated from soybean, exhibited the prominent property of being less bitter than other enzymatic hydrolysates. In the 1st experiment in this series, angiotensin I-converting enzyme (ACE) inhibitory peptides from soy protein hydrolysate by D3 were identified by the establishment of a novel and effective peptide identification method. The amino acid sequences of candidate ACE inhibitory peptides were determined by electrospray ionization mass/mass spectrometry (MS/MS) analysis after rough purification of the samples with gel filtration chromatography and reverse-phase chromatography. Some of the candidate peptides had amino acid sequences that showed homology with those of the reported ACE inhibitory peptides. Then, 8 types of novel candidate peptides were synthesized according to a solid-phase method, and their ACE inhibitory activity was confirmed as the IC₅₀ value. The most potent inhibitor was NWGPLV (IC₅₀= 21 μ M ). In the 2nd experiment, the antihypertensive activity of protein hydrolysates by D3 was investigated in spontaneously hypertensive model rats (SHRs). The dose-dependent antihypertensive effect of soy protein hydrolysate was confirmed, and systolic blood pressure was significantly reduced after the oral administration of doses exceeding 100 mg/kg. Casein hydrolysate was found to have the most potent effects on suppressing blood pressure as well as ACE inhibitory activity among the various food protein hydrolysates studied because of the primary structure of casein. These results indicate that hydrolysates by D3 could be a useful food ingredient because it has the physiological function (antihypertensive activity).     

蛋白质酶解物苦味形成机理及控制研究

蛋白质酶解后产生不同程度苦味主要是由产物中含疏水性氨基酸低分子肽所形成,这些苦味严重影响蛋白质水解产物在食品工业中应用。该文综述不同食物蛋白质酶解物中苦味肽形成机理及蛋白质酶解物苦味的降低、去除及控制方法研究最新进展,主要包括选择性分离、苦味掩盖、酶法及微生物脱苦等。     

扇贝裙边酶解过程中呈味组分的变化规律研究

本研究以扇贝裙边为原料,利用复合蛋白酶对扇贝裙边进行酶解,在确定扇贝裙边最佳酶解温度的基础上,探索了扇贝裙边酶解液在不同酶解时间下的呈味特点,并探讨了不同酶解液中呈味分子的变化规律,探明不同酶解时间下扇贝裙边酶解液的呈味规律。结果表明不同酶解时间制备酶解液的滋味存在显著性差异,其中8 h的扇贝裙边酶解液鲜味强度最高(9.32分),而12 h酶解液的苦味(7.33分)和饱满度(8.33分)最强。主要是因为酶解时间对酶解液鲜味氨基酸和苦味氨基酸的含量及比例存在较大影响,当酶解8 h时,扇贝裙边酶解液中鲜味氨基酸比例最高(46.80%),而苦味氨基酸比例最低(51.67%);此外,肽分子分布结果显示8h酶解液中5000 u的肽段(对呈味贡献小)和180 u的肽段(苦涩味明显)比例较低,可能是8 h扇贝裙边酶解液取较好的鲜味和饱满度,较低苦味的主要原因。本研究通过研究扇贝呈味组分在酶解过程中的变化规律,为工业上利用扇贝裙边制备高品质呈味基料提供理论基础和指导。     

Valorisation of protein hydrolysates from animal by-products: perspectives on bitter taste and debittering methods: a review

Conversion of animal by-products to high value-added food ingredients is one of the top trends in the slaughter industry. Enzymatic hydrolysis of animal by-products can generate protein hydrolysates, which provides an opportunity for effective utilisation. However, bitterness of protein hydrolysate is a major undesirable aspect for various applications. In this review, the current knowledge on protein hydrolysates from animal by-products is briefly reviewed. The structural features of bitter peptides and bitter taste receptors are summarised. Moreover, the potential approaches for debittering protein hydrolysates are highlighted, including exopeptidase treatment, Maillard reaction, plastein reaction and encapsulation. In addition, the current debittering strategies and challenges are also discussed. This article presents some opportunities to utilise protein hydrolysates from animal by-products and their debittering methods.     

蛋白酶处理对脱脂乳感官品质及游离氨基酸的影响

以新鲜脱脂牛乳为原料,采用分光测色仪、电子舌及氨基酸自动分析仪等分析酶解处理对脱脂牛乳感官品质、游离氨基酸含量及组成的影响。结果表明：经风味蛋白酶处理的脱脂牛乳水解度高,达到24.61%;蛋白酶处理会导致其感官性状的改变,与脱脂牛乳相比,3?种酶解产物L*值均显著（P＜0.05）下降,a*（负值）显著上升（P＜0.05）,风味蛋白酶处理对脱脂牛乳色泽影响大于碱性蛋白酶、复合蛋白酶处理;不同酶解产物滋味轮廓之间存在较大差异,与脱脂牛乳相比其甜味值下降显著,且随酶解时间延长,苦味值上升,甜味值衰退,碱性蛋白酶处理的酶解产物以涩味及涩味回味为主,风味蛋白酶的酶解产物以咸、苦味及苦味回味为主,复合蛋白酶的酶解产物以酸味为主,苦涩等味觉较低,电子舌能较好地区分不同酶解物的滋味差异;酶解处理可使脱脂牛乳中的游离氨基酸及必需氨基酸含量显著增加,苦味氨基酸为主要呈味氨基酸。酶解处理及酶解进程会使脱脂牛乳色泽、滋味及游离氨基酸产生变化,其中风味蛋白酶处理产生的影响大于碱性蛋白酶和复合蛋白酶处理。     

Exopeptidases and their application to reduce bitterness in food: a review

When exopeptidases catalyze hydrolysis of peptide bonds, the product(s) may have a less bitter taste, and the free amino acids or small peptides formed may function in food as pleasant-tasting flavor compounds or as flavor precursors. There are several classes of exopeptidase based on specificity for hydrolysis of synthetic substrates. Exopeptidases in food-stuff may be of natural origin or may be extrinsic, that is, produced by microorganisms or parasites. Exopeptidases used to modify foods are also becoming increasingly available in the industrial enzyme market. Exopeptidases contribute to a variety of quality changes in postharvest fruit, meats, and food fermentations. Foodstuff impacted by these enzymes during processing include cocoa, beer, aged and cured meat products, koji, fish sauce, ripened cheeses, and protein hydrolysates. An important role of exopeptidases in food is the hydrolysis of hydrophobic, bitter peptides. The relationship between peptide structure and sensory transduction/receptor models is discussed. Research on the use of exopeptidases to reduce bitterness is reviewed.     

Enzymatic Production,Bioactivity, and Bitterness of Chickpea (Cicer arietinum) Peptides

Chickpeas are inexpensive, protein rich (approximately 20% dry mass) pulses available worldwide whose consumption has been correlated with positive health outcomes. Dietary peptides are important molecules derived from dietary proteins, but a comprehensive analysis of the peptides that can be produced from chickpea proteins is missing in the literature. This review provides information from the past 20 years on the enzymatic production of peptides from chickpea proteins, the reported bioactivities of chickpea protein hydrolysates and peptides, and the potential bitterness of chickpea peptides in food products. Chickpea peptides have been enzymatically produced with pepsin, trypsin, chymotrypsin, alcalase, flavorzyme, and papain either alone or in combination, but the sequences of many of the peptides in chickpea protein hydrolysates remain unknown. In addition, a theoretical hydrolysis of chickpea legumin by stem bromelain and ficin was performed by the authors to highlight the potential use of these enzymes to produce bioactive chickpea peptides. Antioxidant activity, hypocholesterolemic, and angiotensin 1‐converting enzyme inhibition are the most studied bioactivities of chickpea protein hydrolysates and peptides, but anticarcinogenic, antimicrobial, and anti‐inflammatory effects have also been reported for chickpea protein hydrolysates and peptides. Chickpea bioactive peptides are not currently commercialized, but their bitterness could be a major impediment to their incorporation in food products. Use of flavorzyme in the production of chickpea protein hydrolysates has been proposed to decrease their bitterness. Future research should focus on the optimization of chickpea bioactive peptide enzymatic production, studying the bioactivity of chickpea peptides in humans, and systematically analyzing chickpea peptide bitterness.     

The improving effect of spray-drying encapsulation process on the bitter taste and stability of whey protein hydrolysate

Although whey protein hydrolysate (WPH) possesses good physiological functionality, its bitter taste and hygroscopic property limit its direct utilization as food ingredient. The aim of this work was to encapsulate whey protein hydrolysate by spray drying using maltodextrin or maltodextrin/??-cyclodextrin mixture as wall materials to attenuate the bitter taste and enhance the stability of whey protein hydrolysate. Hygroscopicity, glass transition temperature, bitter taste, and morphology of non-encapsulated WPH and encapsulated WPH were evaluated. Solubility, particle size, bulk density, and moisture content were also measured. Compared with the non-encapsulated WPH, the encapsulated WPH exhibited significantly lower hygroscopicity and higher glass transition temperature. The bitterness of both maltodextrin-encapsulated WPH and maltodextrin/??-cyclodextrin-encapsulated WPH was significantly lower than that of the original non-encapsulated WPH. Morphological analysis by scanning electron microscopy showed that the microcapsules of the spray-dried encapsulated WPH were matrix-type with less link bridge and had a continuous wall with many concavities. In addition, encapsulation process did not exert negative effect on the solubility of whey protein hydrolysate. The results indicated that encapsulation with maltodextrin and ??-cyclodextrin as carriers was helpful to attenuate the bitter taste and enhance the stability of whey protein hydrolysate.     

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates

ABSTRACT:  The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzyme™ produced protein hydrolysates with significantly higher ( P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (≤ 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly ( P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher ( P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and α-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and α-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.     

食品外肽酶的研究进展

外肽酶包括氨肽酶和羧肽酶两大类。当外肽酶催化多肽水解时,产物苦味减轻,同时生成的游离氨基酸和小肽类物质能够形成食品良好风味,或作为风味前体物质。外肽酶能够在食品后熟阶段起作用,如水果成熟、种子萌发和肉类陈化,也能够在食品加工处理过程中起作用,如干燥、热处理和发酵,从而决定终产品品质的优劣。外肽酶在食品中的重要应用主要体现在能够水解疏水性苦味肽,并去除苦味。本文较为系统地综述近年来外肽酶及其在食品中的研究现状,主要包括外肽酶的分类、结构、反应特性,以及在食品加工过程中对蛋白质水解产物的最终产品良好风味的贡献机制。     

实用类蛋白反应的作用机制及其对海洋源蛋白修饰的研究进展

类蛋白反应作为一种可以修饰生物活性肽的新方法,已经成为蛋白质食品研究的热点。海洋生物含有丰富的生物活性成分,其含有的蛋白肽具有多种人体代谢和生理调节功能。然而蛋白肽存在生物利用度有限、酶解液味苦等问题。本文在阐述类蛋白反应过程和机制的基础上,对类蛋白反应修饰提高海洋源活性蛋白肽的生物活性、改善蛋白的加工特性及减少蛋白酶解液苦味等研究进展进行了详细的介绍,以期为海洋源蛋白的深度利用以及高值化研究提供理论参考。     

Hydrolysis of casein: a comparative study of two proteases and their peptide maps

Gel electrophoresis (SDS-PAGE) and reversed phase-high performance liquid chromatography (RP-HPLC) techniques were used to illustrate the actions of two enzymes, a pineapple stem bromelain and a Bacillus protease ( Bacillus subtilis ), on bovine caseins. the aim of the study was to ascertain the possible future application of the enzymes in the production of bitter peptides.
The results illustrate that the individual actions of the two proteases produce hydrolysates of very different peptide composition. the Bacillus protease action on casein results in the production of a greater variety of hydrophobic, low molecular weight (<10 kDa) peptides, due to a more extensive proteinase activity. Bromelain action however results in a hydrophobic hydrolysate containing a greater number of high molecular weight peptides (>10 kDa), which are not normally associated with flavour fractions.
The results of the study, show that the hydrolysate produced by the Bacillus protease action on casein, is a greater potential source of bitter peptides for future study, than the hydrolysate produced by bromelain.     

大豆蛋白水解物苦味评价方法

目的:研究大豆蛋白水解物的苦味程度,寻找苦味评价方法.方法:采用感官评价和电子舌分析大豆蛋白水解物的苦味程度;采用高效液相色谱法分析游离氨基酸含量和肽分子质量分布;采用偏最小二乘回归分析(partial least squares regression,PLSR)研究游离氨基酸含量、感官苦味强度以及电子舌苦味响应值评价...     

大豆蛋白水解液脱苦的研究

大豆蛋白酶解常常会产生苦味,蛋白质水解物苦味肽的苦味是长期困扰其应用的问题。本文研究了酶法与微生物法对大豆蛋白水解液脱苦的效果。结果表明:采用端肽酶黑曲霉酸性蛋白酶(3000u/g)与内切酶枯草杆菌碱性蛋白酶(Alcalase 2.4L)协同作用水解大豆蛋白可有效降低水解液苦味,并且由酿酒酵母对水解液进一步处理后,大豆蛋白水解液的苦味降至更低。     

Bitterness intensity of soybean protein hydrolysates—chemical and organoleptic characterization

Soluble and isoelectric soluble soybean protein hydrolysates were prepared by Alcalase treatment to a degree of hydrolysis of 3–15%. The bitterness intensity of the hydrolysates obtained was assessed on a five-point scale. The average relative molecular masses of peptides in the soluble hydrolysates (2250-1400) and isoelectric soluble hydrolysates (1313-800) and their hydrophobic peptide fractions (575-400) were determined by the trinitrobenzenesulphonic acid method. The molecular mass distribution of peptides in soluble and isoelectric soluble soybean hydrolysates and their hydrophobic peptide fractions was determined by gel permeation HPLC using a Zorbax Bio Series GF-250 column. The results suggest that the main reason for the bitterness of soybean protein hydrolysates prepared by Alcalase treatment are hydrophobic bitter peptides of relative molecular mass less than 1000.     

The use of spray drying technology to reduce bitter taste of casein hydrolysate

The utilization of protein hydrolysates in food systems is frequently hindered due to their bitterness and hygroscopicity. Spray drying technology could be an alternative for reducing these problems. The aim of this work was to reduce or to mask the casein hydrolysate bitter taste using spray drying and mixtures of gelatin and soy protein isolate (SPI) as carriers. Six formulations were studied: three with 20% of hydrolysate and 80% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%) and three with 30% of hydrolysate and 70% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%). The spray-dried formulations were evaluated by SEM, hygroscopicity, thermal behavior (DSC), dissolution, and bitter taste, by a trained sensory panel using a paired-comparison test (free samples vs. spray-dried samples); all samples were presented in powder form. SEM analysis showed mostly spherically shaped particles, with many concavities and some particles with pores. All formulations were oil and water compatible and showed lower hygroscopicity values than free casein hydrolysate. At Aw 0.83, the free hydrolysate showed Tg about 25 °C lower than the formulations, indicating that the formulations may be more stable at Aw ≥ 0.65 since the glass transition should be prevented. The sensory panel found the formulations, tasted in the powder form, to be less bitter (P < 0.05) than the free casein hydrolysate. These results indicated that spray drying of casein hydrolysate with mixtures of gelatin and SPI was successful to attenuate the bitterness of casein hydrolysate. Thus, spray drying widens the possibilities of application of casein hydrolysates.     

Original article: Thermal pretreatment and chemical modifications as a means to alter hydrolytic characteristics and prevent bitterness in hydrolysates of fishery by‐catch (Decapterus maruadsi) protein

In this study, fishery by‐catch protein (Decapterus maruadsi) was first thermal treated (60–100 °C), phosphorylated (0.5–4.0 g g^?1 protein) or succinylated (0.05–0.3 g g^?1 protein) before hydrolysis to prevent the bitterness of the resulting hydrolysate. Hydrolysis was performed at 50 °C, 10 h, pH 8.0 by Alcalase. Results showed that thermal treatment upon 80 °C, succinylation and phosphorylation resulted in significant decrease in amino acid content and increase in larger peptide content (>1450 Da). Succinylation did not reduce the bitterness as a result of higher content of small peptides (<550 Da), which was found to be correlated positively with the bitterness of hydrolysates. However, thermal treatment, especially phosphorylation, could reduce the bitterness to discernible level, indicating that phosphorylation before hydrolysis could be a potential means to prevent the evolution of bitter flavour which restricts the practical uses of the hydrolysates.