Umami and umami-enhancing peptides from myofibrillar protein hydrolysates in low-sodium dry-cured Spanish mackerel (Scomberomorus niphonius) under the action of Lactobacillus plantarum

Lactobacillus plantarum was involved in producing low-salt (2%, w/w) dry-cured Spanish mackerel, and its effect on the degradation of myofibrillar protein and production of umami peptides was investigated. Results showed that the dominant microbiome of the sample was Lactobacillus plantarum, accounting for 95.7% of the microbial community. The umami peptides in this dry-cured mackerel were extracted and separated by ultra-filtration and gel filtration chromatography and further identified by Nano-LC–MS/MS. Five umami-enhancing peptides, including TFYNELRV (1040 Da), TFYNELR (941 Da), TLFQPSF (838 Da), LYANNVL (805 Da) and FAGDDAPRAVFPS (1348 Da), were identified to be derived from myofibrillar protein, and the percentage of their hydrophobic amino acid were 42.85%, 33.33%, 57.14%, 57.14% and 53.84%, respectively. The synthetic peptides of these five umami peptides showed an umami-enhancing threshold value ranging from 0.34 to 0.45 mg mL⁻¹ through sensory evaluation. The SDS-PAGE analysis exhibited that actin band intensity was decreased during the processing of dry-cured mackerel. Therefore, Lactobacillus plantarum has the potential to improve the hydrolysis of myofibrillar protein to produce umami peptides in dry-cured mackerel.     

酱油中鲜味二肽的分离鉴定及其呈味特性研究

为了明确酱油中鲜味肽的结构序列,本文对酱油中鲜味肽进行分离鉴定,并系统地研究了其呈味特性。酱油经超滤膜(膜通量:5 ku、3 ku和1 ku)分离获得4个组分(F1,F2,F3和F4),通过感官评定筛选出鲜味最强的组分F4(1 ku)。该组分通过Sephadex G-15凝胶层析色谱继续分离得到8个组分(P1、P2、P3、P4、P5、P6、P7和P8),经感官评定筛选出鲜味最强的组分P2。采用超高压液相色谱串联质谱技术对P2组分进行多肽结构鉴定,通过手动De novo测序得到4条新的鲜味肽,其序列分别是Asn-Pro(230.1135 u)、Ala-His(227.1026 u)、Gly-Pro(173.0929 u)和Gly-Leu(189.1230 u)。然后采用固相合成技术合成四条肽并通过感官评定和电子舌分析,结果表明四条肽均具有明显的鲜味或鲜味增强作用。进一步研究发现,酱油的鲜味不仅来自于谷氨酸和天冬氨酸等鲜味氨基酸,小分子肽类也是构成酱油鲜味的重要成分之一。     

不同底物浓度花生粕酶解产物特性的研究

花生粕是花生榨油后的副产物,压榨过程中高度变性导致蛋白利用率较低。本文通过生物发酵技术诱导米曲霉分泌蛋白酶水解花生粕,在不同底物浓度条件下酶解花生粕,对比酶解产物的蛋白回收率、水解度、总糖回收率、分子量分布、游离氨基酸组成,同时采用电子舌对富含呈味肽的酶解产物进行感官评价分析,探究不同底物浓度条件下发酵花生粕的酶解产物特性,并筛选适宜的酶解浓度。研究发现,发酵花生粕经不同底物浓度酶解后,蛋白回收率在70.68%~84.14%之间,水解度在30.04%~40.05%之间;酶解产物以小于1 ku的短肽为主(87.57%~90.21%),并含有较多游离氨基酸;在呈味特性方面,酶解液含有较强的鲜味。结果表明,在料液比为1:5时,获得较高的蛋白回收率、水解度以及较多小分子肽,鲜味评分也相对较高。     

白玉菇中鲜味肽的分离鉴定及呈味特性分析

采用超滤、凝胶渗透色谱及反相高效液相色谱结合感官引导,从白玉菇水提物中分离纯化出白玉菇鲜味最佳组分,通过超高效液相色谱-质谱联用技术从中鉴定鲜味肽,并将所鉴定的鲜味肽合成后通过人体感官评价对其呈味特性进行研究。结果表明：白玉菇水提物中含有6?种鲜味肽,即ELELQ、ELQSGNTY、NYNGGY、EAKVY、VANGGGFGAA、SLLQPL,鲜味阈值分别为0.16、0.19、0.31、0.38、0.44、0.63?mg/mL,且6?种肽的分子质量皆在1?000?Da以下;同时6?种合成肽具有鲜味增强作用,从其在味精溶液中的剂量-反应曲线可以看出,所鉴定的鲜味肽增鲜能力差异显著（P＜0.05）,其中NYNGGY、ELELQ的增鲜能力突出;6?种合成肽对鸡汤具有鲜味、咸味和甜味增强特性,其中ELELQ、NYNGGY、ELQSGNTY对鸡汤的味感提升效果最好。     

Characterization of antioxidant activity and volatile compounds of Maillard reaction products derived from different peptide fractions of peanut hydrolysate

Five peptide fractions isolated from peanut hydrolysate were purified by ultrafiltration and gel filtration chromatography. Their reaction degrees with glucose or lecithin in Maillard reaction were compared. Glucose consumption, oxygen radical absorbance capacity (ORAC) values and volatile compounds composition of peanut hydrolysate (PH) and its peptide fractions after thermal treatment and Maillard reaction were evaluated. Results revealed that the peptide fraction of 1-3 kDa showed the highest antioxidant activity. The peptides with smaller molecular weight showed a higher reaction degree to increase antioxidant activity and to produce more volatile compounds through Maillard reaction. Analysis of molecular weight distribution showed that peptide degradation and cross-linking simultaneously occurred during the Maillard reaction. In addition, lecithin addition to peptide-glucose system caused a significant increase of antioxidant activity possibly due to more nitrogen-containing compounds formed.     

不同结构改性与美拉德反应前后鲜味二肽(Asn-Pro和Ala-His)呈味特性的对比分析

本文为了探讨不同结构改性(氨基酸顺序改变、增加鲜味氨基酸、串联肽)与美拉德反应对鲜味二肽(Asn-Pro和Ala-His)鲜味与增鲜效果的影响,利用电子舌分析了Asn-Pro和Ala-His及其结构改性后获得的新肽和美拉德反应产物的呈味特性,评价了Asn-Pro和Ala-His结构改性前后以及美拉德反应对味精(MSG)和酱油的增鲜效果。结果表明,Ala-His序列改性后获得的新二肽His-Ala的鲜味评分由4.09分上升到5.08分,对MSG和酱油具有明显的增鲜作用,其鲜味评分分别提高了1.40分和0.48分,而其他结构改性获得的9条新肽(Pro-Asn、Glu-Asn-Pro、Asn-Pro-Glu、Asp-Asn-Pro、Asn-Pro-Asp、Asn-Pro-Asn-Pro、Ala-His-Ala-His、Asn-Pro-Ala-His和Ala-His-Asn-Pro)相对于原二肽的鲜味均有所下降,对MSG和酱油具有明显的鲜味抑制作用。美拉德反应可显著提升Asn-Pro和Ala-His的鲜味特性,其鲜味评分分别由7.65分和4.09分上升到9.48分和6.37分,美拉德反应产物对MSG和酱油具有明显的增鲜作用,MSG的鲜味评分分别提高了3.96分和2.29分,酱油的鲜味评分分别提高了3.77分和1.49分。氨基酸序列改变、增加鲜味氨基酸以及串联肽能显著影响多肽鲜味及增鲜效果,美拉德反应是提升多肽鲜味与增鲜效果的有效途径。     

不同鲜味物质对干腌马鲛鱼鲜味的贡献与比较分析

以8?种市售干腌马鲛鱼为研究对象,通过比较不同产品中鲜味物质成分和含量差异,并结合其滋味强度值和味精当量的分析计算,以确定对干腌马鲛鱼鲜味贡献最大的物质。结果表明：干腌马鲛鱼具有突出的咸、鲜味和浓厚味感,而其鲜味主要是由谷氨酸、鲜味肽等鲜味物质和乳酸等增鲜物质赋予。在其鲜味物质中,谷氨酸的滋味强度值大于1（除3、5、7号样品）,显示出它对干腌马鲛鱼鲜味的突出贡献,超滤组分的感官分析和肽分子质量分布测定结果表明,分子质量小于1?000?Da的小分子肽对样品的鲜味具有重要贡献。此外,长时间的风干可能会促进乳酸和鲜味肽的生成,但随着水分的流失,其苦味和咸味也增高;而辅料蔗糖和白酒的添加将抑制乳酸的产生,且蔗糖能在一定程度上促进谷氨酸的生成。值得强调的是,8?种样品的核苷酸均已降解为肌苷（inosine,HxR）和次黄嘌呤（hypoxanthine,Hx）等苦味物质,且长时间的风干可能会促进HxR和Hx的积累。     

鲜味肽与增鲜肽及其加工特性研究进展

鲜味肽与增鲜肽是食品中的关键滋味成分,具有风味突出等优势。同时,鲜味肽与增鲜肽种类复杂且常作为反应前体物影响食品的风味特性,对食品风味有重要影响。据统计分析,目前已报道鲜味肽与增鲜肽数量分别为208条和60条,鲜味肽氨基酸组成和空间结构是决定鲜味肽鲜味特性的重要因素,而增鲜肽具有更复杂的增鲜模式和构效关系。在食品加工过程中,鲜味肽与增鲜肽会作为活泼反应底物参与酶促与非酶促反应,并在一定条件下转变为鲜味更强或增鲜特性更优的反应产物,有利于食品风味品质的提升。作者系统分析了鲜味肽与增鲜肽的结构特性,探讨了基于不同加工方式其在食品加工过程中的变化及其对食品呈味特性的有益影响,并阐述了在研究过程中存在的问题和解决方案,旨在对鲜味科学研究及调味品行业的技术发展提供理论指导。     

Purification and identification of kokumi-enhancing peptides from chicken protein hydrolysate

The Maillard reaction products (MRPs) from chicken protein hydrolysate were demonstrated to have intense umami and kokumi-enhancing effects. To find the main flavour-enhancing compounds in the chicken protein hydrolysate, the fractions with different molecular weights were obtained by ultrafitration. The evaluation of taste characteristics revealed that the fractions with molecular weights ranging from 1000 to 5000 Da predominantly contributed to the umami and kokumi-enhancing effects. After further purification by using ultrafiltration, gel filtration chromatography, and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in combination with sensory evaluation, three peptides were identified, an octapeptide (WVNEEDHL), a nonapeptide (NSLEGEFKG) and a decapeptide (KDLFDPVIQD). Sensory evaluation results showed that all three peptides could significantly enhance the meat flavour, umami taste and thickness of the chicken powder solution. The results indicate that the peptides have potential application as effective chicken flavour-enhancing ingredients in the food industry.     

Evaluation of Peptide Contribution to the Intense Umami Taste of Japanese Soy Sauces

ABSTRACT: Koikuchi and tamari shoyu are 2 types of traditional Japanese soy sauce whose low-molecular-weight fractions (less than 500 Da) are known to have the most intense umami taste. Given that peptides were also abundant in some umami fractions of both shoyu, further investigation was conducted to evaluate the precise contribution of peptides to the soy sauce tastes. To locate and isolate peptide fractions, separation procedures using Sephadex G-25 SF, Sephadex G-10, and reversed-phase high-performance liquid chromatography (RP-HPLC) linked to sensory evaluation (by taste dilution analysis), as well as amino acid analysis were performed. Peptides having glutamyl residue were present in most peptide fractions. However, they all revealed negligible contributions to the intense umami taste of the soy sauces. On the other hand, free Glu, in the presence of sodium salt, as well as free Asp and several sweet-taste eliciting free amino acids were considered to be the main contributors to the umami taste of the soy sauces. This study of Japanese soy sauces gives a clear explanation of the frequently questioned peptide contribution to the taste of savory food product.     

风干武昌鱼中鲜味肽的分离纯化及二级结构分析

采用电子舌测定鲜味,超纳滤、反相高效液相色谱从风干武昌鱼中分离纯化出纯度均一的鲜味肽,基质辅助激光解析电离-飞行时间质谱仪、氨基酸自动分析仪、傅里叶变换红外光谱测定鲜味肽的分子质量、氨基酸组成和二级结构,探讨鲜味肽结构与风味的关系。结果显示：从风干武昌鱼中纯化得到了一种鲜味较强的肽P2a-2,其分子质量为1 304.59 Da。P2a-2的鲜味氨基酸谷氨酸（21.97%）、甘氨酸（10.67%）和丙氨酸（9.51%）比例较高,且助味氨基酸组氨酸的比例最高（27.54%）,β-转角为主要的二级结构（90.16%）,推断P2a-2呈现较佳鲜味与其高含量的鲜味氨基酸和β-转角相关。     

谷朊粉发酵液中鲜味肽的分离、鉴定与呈味分析

鲜味是五种基本味感之一,它在人们选择食品中扮演了越来越重要的角色,肽是氨基酸组成的混合物,具有多种味道,是主要的呈味物质,利用蛋白酶解可以获得具有较强鲜味的肽段。本实验采用感官评价结合超滤(UF)、凝胶层析(GPC)和反相高效液相色谱(RP-HPLC)等技术从以盐酸脱酰胺面筋蛋白为原料的发酵液中分离纯化得到一种鲜味肽,同时采用MALDI-TOF-MS串联质谱鉴定该鲜味肽的氨基酸序列及其分子质量;目标定向固相合成鉴别出的鲜味肽,并测定了其鲜味阈值及鲜味相乘效应。结果发现:该鲜味肽的氨基酸序列为Asp-Cys-Gly(DCG),分子质量为293.15 Da,固相合成该鲜味肽后采用滋味稀释法评价发现该鲜味肽的鲜味阈值为100 mg/m L,是MSG阈值的1/3,该鲜味肽对浓度为200 mg/L的I+G溶液具有鲜味增强作用,对MSG没有鲜味增强作用。     

养殖暗纹东方鲀肌肉中呈味肽的分离鉴定

本文旨在从生鲜暗纹东方鲀肌肉中提取呈味肽从而对暗纹东方鲀肌肉中的呈味肽的结构特性进行相应研究。将生鲜养殖暗纹东方鲀肌肉水提物经Sephadex G15凝胶柱分离,得到3个多肽组分(分别为0-F1、0-F2和0-F3),感官评价结合电子舌选择出具有最强烈的鲜味和kokumi感的组分0-F1。采用反相高效液相色谱(RP-HPLC)对其进行进一步分离得到2个组分0-F1-1和0-F1-2,电子舌鉴定显示0-F1-1组分有较强的鲜味。最后利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对RP-HPLC分离得到组分0-F1-1中呈味肽氨基酸序列进行测定,鉴定得到一种新的呈味七肽结构:Pro-A-Ala-B-Met*-Cys-Arg(810.9046 Da,A和B均为氨基酸代码,Met*表示氧化型甲硫氨酸)虽然肽段中含有大量的疏水性氨基酸,但该肽不具有苦味,而且有显著的浓厚感,其中含有的Cys残基可能是其具有kokumi感的关键。     

养殖暗纹东方纯肌肉中呈味肽的分离鉴定

本文旨在从生鲜暗纹东方纯肌肉中提取呈味肽从而对暗纹东方纯肌肉中的呈味肽的结构特性进行相应研究。将生鲜养殖暗纹东方鱼屯肌肉水提物经Sephadex G15凝胶柱分离,得到3个多肽组分（分别为0-F1、0-F2和0-F3）,感官评价结合电子舌选择出具有最强烈的鲜味和kokumi感的组分0-F1。采用反相高效液相色谱（RP-HPLC）对其进行进一步分离得到2个组分0．F1．1和0．F1．2,电子舌鉴定显示O．F1．1组分有较强的鲜味。最后利用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）对RP-HPLC分离得到组分0-F1-1中呈味肽氨基酸序列进行测定,鉴定得到一种新的呈味七肽结构：Pro-A-A1a-B-Met＊-Cys-Arg（810．9046 Da,A和B均为氨基酸代码,Met＊表示氧化型甲硫氨酸）虽然肽段中含有大量的疏水性氨基酸,但该肽不具有苦味,而且有显著的浓厚感,其中含有的Cys残基可能是其具有kokumi感的关键。     

Sensory Characteristics and Antioxidant Activities of Maillard Reaction Products from Soy Protein Hydrolysates with Different Molecular Weight Distribution

Soy protein was hydrolyzed using two enzymes to obtain soy protein hydrolysate (SPH), the SPH was fractionated with ultrafiltration membranes to obtain peptide fractions below 1,000 Da (SP1) and 1,000–5,000 Da (SP2), and for the meantime, SPH was further completely hydrolyzed to get compound amino acids (CAA). Maillard reaction products (MRPs) were prepared from aqueous xylose–SPH/SP1/SP2/CAA model systems by heating at 120°C for 2.0 h. Compared with the original hydrolysates and other MRPs, the MRPs from SP2 exhibited a distinctly enhanced effect on flavor, including the caramel-like, soy sauce-like odors, umami and mouthful tastes and a greatly reduced bitterness in consomme′ soup. Antioxidant activities of SPH, SP1, SP2, CAA, and their MRPs were investigated through reducing power, DPPH radical-scavenging activity, and Fe²⁺ chelating activity. Before Maillard reaction, the antioxidant activities of peptide fractions with different molecular weights were quite different, and SP2 showed the highest activity; however, CAA exhibited very poor antioxidant activity. The antioxidant activities of SPH, SP1, SP2, and CAA were greatly enhanced by Maillard reaction, and the MRPs prepared from xylose–CAA model system exhibited a higher antioxidant activity than those from other model systems. Pyrazines, pyrroles, furans, and thiazoles were significantly correlated with reducing power and Fe²⁺ chelating activity by principal component analysis.     

养殖暗纹东方鲀中7 种醇提鲜味肽的鉴定及其呈鲜特性

为进一步发掘鲜味肽并探索其构效关系,以60%乙醇溶液作为提取剂,采用超滤纳滤、凝胶色谱层析并结合人工感官评价,从养殖暗纹东方鲀肌肉中分离纯化多肽馏分,进一步利用纳升液相四极杆轨道阱质谱鉴定多肽的分子质量及氨基酸组成。将初步鉴定肽进行固相合成,结合人工感官及电子舌技术验证其呈味特性,并采用分子对接探讨多肽结构与鲜味的关系。结果表明：7 条新鉴定的鲜味肽NWDDMEK、KTGLSPDQF、KTDLNFENL、ASLDGEFKG、ALASLDGEFKG、ALTSLDGEFKG和RLGSSEVEQVQ,其分子质量范围为922.3～1 230.6 Da,鲜味阈值范围为0.38～1.04 mmol/L。鲜味肽序列上的谷氨酸（Glu）、天冬氨酸（Asp）和赖氨酸（Lys）及鲜味受体上的N69、S276、R151、R277、A302、T149和N150等氨基酸残基对多肽的呈鲜特性有重要的意义。本研究可为后期进一步探明肽的呈鲜规律提供参考。     

面筋蛋白咸味肽的分离纯化及结构鉴定

为了明确面筋蛋白酶解液中的咸味肽序列，对面筋蛋白咸味酶解物进行了分离纯化和结构鉴定。对酶解物脱盐处理后进行分离纯化。经过分级超滤，选择咸味最高、分子质量1 000 Da以下的组分进行葡聚糖凝胶Sephadex G-15过滤层析，结合感官评定结果筛选出咸味最强的组分，并利用液相色谱-串联质谱鉴定其氨基酸序列。最终得到面筋蛋白中咸味肽氨基酸序列为PFGQQ、PFSPQ、QPFP、PDFP、FDDP，分子质量分别为576.28、575.28、488.25、475.22、493.21 Da。本研究为面筋蛋白咸味肽的开发提供了一定理论依据。     

武定鸡肉中鲜味肽分离鉴定及呈味特性

利用超滤分级、凝胶过滤色谱法和反相-高效液相色谱法对武定鸡肉中鲜味肽进行分离纯化,结合感官评价追踪鲜味最强烈的组分,以纳升高效液相色谱-串联质谱联用技术与固相合成法对鲜味肽进行鉴定和合成,进一步分析鲜味肽的序列来源及其呈味特征。结果表明：从武定鸡肉中分离鉴定出8 条多肽,分子质量范围为365.20～1 735.92 Da,其中LDF、FVT和DLAGRDLTDYLMKIL这3 条肽段具有明显的鲜味活性,阈值在0.062～0.250 mg/mL之间,是武定鸡肉鲜味的关键组分。     

Isolation and identification of flavour peptides from Puffer fish (Takifugu obscurus) muscle using an electronic tongue and MALDI-TOF/TOF MS/MS

To clarify the key flavour peptides that account for the cooked taste of puffer fish, this study was performed to examine flavour peptides extracted from the flesh of puffer fish (Takifugu obscurus). Peptides fractions (P1, P2, P3, P4 and P5) were purified from an aqueous extract of T. obscurus muscle by ultrafiltration and Sephadex G-15 gel filtration chromatography (GFC). P2 was further fractionated into P2a, P2b, and P2c by reverse phase high performance liquid chromatography (RP-HPLC). Fraction P2b elicited umami and sweet taste. The amino acid sequence of P2b subfraction was identified as Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val (836.4Da) by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF/TOF MS/MS). Hydrophilic amino acids residues Tyr, Gly, Gly, Thr, and Phe are likely to contribute to the umami and sweet taste of this octapeptide. The results of this study suggest this peptide is one of important components of the 'mellowness' and 'tenderness' taste of the T. obscurus.     

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.