In vitro digestion and characterisation of 2S albumin and digestion‐resistant peptides in pecan

The 2S albumins are one of the major protein families involved in severe food allergic reactions to nuts, seeds and legumes, thus potentially making these proteins clinically relevant for allergic sensitisation and potential diagnostic markers. In this study, we sought to purify native 2S albumin protein from pecan to further characterise this putative allergen. The purified 2S albumin, Car i 1, from pecan was found to be resistant to digestion by pepsin in simulated gastric fluid (SGF) and comparatively stable to proteolysis by trypsin and pancreatin in simulated intestinal fluid (SIF). Digestion of purified Car i 1 in SGF and SIF resulted in formation of different digestion‐resistant peptides that were capable of binding IgE antibodies from allergic individuals. Digestion stability of Car i 1 and formation of digestion‐resistant antigenic peptides may explain why it is a potent sensitising protein in pecan for susceptible individuals. The observation that digestion‐resistant peptides are able to bind IgE implies that pecan can trigger systemic allergic reactions even after digestion in the stomach and small intestine.     

Identification of hemocyanin as a novel non-cross-reactive allergen from the giant freshwater shrimp Macrobrachium rosenbergii

Scope : Sensitization to giant freshwater shrimp Macrobrachium rosenbergii (Mr) was recently reported. However, the allergens have yet to be identified. This study aimed to identify and characterize a novel allergen of Mr shrimp. Methods and results: Extracted proteins were separated and purified by anion and in some experiments, size‐exclusion chromatography. Serum IgE from shrimp allergic donors identified a candidate protein, which was characterized by LC‐MS/MS. The specificity of IgE binding was tested using immunoblotting and inhibition ELISA. The IgE‐binding profiles from 12 of 13 Mr allergic subjects that were pre‐incubated with an extract of Penaeus monodon showed residual binding to ～60–80 kDa proteins. The 60–80 kDa IgE‐bound proteins were fractionated in the flow‐through of anion chromatography showing a high IgE reactivity. Peptides identified by LC‐MS/MS showed the proteins closely match subunits of hemocyanin (Hcs). Purified Hcs from hemolymph markedly inhibited binding of IgE from sera of Mr allergic subjects to solid‐phased Mr proteins in inhibition ELISA. Conclusion: Hcs were identified as heat‐stable, non‐cross‐reactive, high‐molecular‐weight (MW) allergens from Mr shrimp. Since circulatory organs are not always removed during food preparation, high concentrations of Hcs may be present along with shrimp meat, which contains the known cross‐reactive tropomyosin protein.     

Evaluation of Antioxidant Activities of Zein Protein Fractions

Zein protein was extracted from the by‐product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC‐ESI‐Q‐TOF MS/MS as Pro‐Phe (263.13 Da) and Leu‐Pro‐Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry.     

桃仁多肽螯合亚铁的结构表征及体外模拟消化

以桃仁蛋白酶解分离所得多肽和氯化亚铁为原料,研究不同品种多肽、铁盐和不同分子质量多肽组分对螯合率的影响,以及对多肽螯合亚铁(PKP3-Fe)的结构表征和体外模拟消化的影响。研究结果表明,桃仁多肽与亚铁离子的螯合率显著高于(P<0.05)大豆多肽、玉米多肽和鱼胶原蛋白肽,氯化亚铁和小分子质量桃仁多肽具有更高的螯合率。桃仁多肽与亚铁离子螯合前、后的紫外光谱和荧光光谱图对比显示,螯合后紫外吸收峰位置、峰值均发生迁移,内源荧光强度明显减弱,有螯合物形成;傅里叶红外光谱分析表明,亚铁离子与桃仁多肽中的-COO-、N-H、C-N、O-H形成配位键;扫描电镜图显示,桃仁多肽螯合后微观结构发生明显改变,有光滑球状颗粒生成。在模拟胃部消化过程中,PKP3-Fe的铁离子释放率显著低于硫酸亚铁片和乳酸亚铁片(P<0.05),避免了大量氢氧化铁沉淀的生成,进入模拟肠液后,PKP3-Fe仍有相当部分成分在肠道中以离子态或与多肽以螯合物的状态存在,能更好地被人体吸收利用。     

酪蛋白磷酸肽副产物中ACEI肽的分离鉴定及稳定性研究

酪蛋白磷酸肽副产物中仍含有丰富的功能性多肽,从中回收具有降压活性的血管紧张素I转化酶抑制肽具有废物利用和环境保护的意义。本文在前期研究富集活性肽工艺的基础上,从粗富集产物中进一步分离纯化出具有降压活性的多肽单体,并对单体的分子量、一级序列及其稳定性进行了研究。研究结果表明,以粗富集产物为原料,仅通过阴离子交换树脂及液相制备两步就可获得活性较高的转化酶抑制肽单体P16和P18。P16分子量742.6,序列为GPFPIIV,属首次发现;P18分子量1385.8,序列为FFVAPFPE VFGK。二者均具有较高的耐酸碱性和热稳定性,经体外模拟胃肠消化过程后,发生不同程度的分解,但P16的活性在分解后反而升高12.8%,P18则降低37.5%。推测活性的升高或降低均与活性中心暴露或破坏有关。     

Extraction and mass spectrometry identification of a major peach allergen Pru p 1

BACKGROUND: Peach allergy can be caused by the allergen Pru p 1. This occurs by cross‐reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)‐binding protein extracted from peach fruit has never been reported. RESULTS: Phosphate‐buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach‐allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two‐dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. CONCLUSION: Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1‐homologous peach allergen Pru p 1. Copyright © 2011 Society of Chemical Industry     

QIGLF, a novel angiotensin I-converting enzyme-inhibitory peptide from egg white protein

BACKGROUND: Novel angiotensin I‐converting enzyme (ACE)‐inhibitory peptides from egg white protein can be rapidly screened by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, several peptides with higher ACE‐inhibitory activity were prepared from egg white protein by enzymatic hydrolysis with Alcalase, purified with Sephadex G25, identified by LC/MS/MS and their structure determined by circular dichroism (CD) spectroscopy. RESULTS: Peptide sequences DHPFLF, HAEIN and QIGLF that showed ACE‐inhibitory activity were investigated further for their stability in gastrointestinal solution and for changes in their secondary structure in solution mixtures. QIGLF exhibited the highest activity (IC₅₀ = 75 µmol L^?1) and was resistant to digestion by proteases of the gastrointestinal tract. The CD spectrum of QIGLF showed the presence of the α‐helix conformation. CONCLUSION: Three peptides were identified by LC/MS/MS and synthesised by Fmoc solid phase synthesis. Of the three, only the peptide sequence QIGLF was a potential ACE inhibitor, with an IC₅₀ value of 75 µmol L^?1. Moreover, QIGLF showed low gastrointestinal enzyme susceptibility and contained a relatively high amount of α‐helix. Copyright © 2011 Society of Chemical Industry     

Preliminary studies on the effect of processing on the IgE reactivity of tomato products

Tomatoes and tomato products, obtained from a market, from companies or prepared in research laboratories, were tested in order to investigate how different processes could modulate the IgE reactivity of these products. The protein fraction of the samples was extracted by using a low‐temperature method in order to avoid degradation, and then separated on SDS PAGE to evaluate the protein profile. All the separated samples were blotted onto nitrocellulose membranes and then individually tested with pooled human sera that had been obtained from patients allergic to tomato, in order to evaluate the potential IgE binding of the protein bands. Heat treatments conventionally employed for the production of tomato‐based products were found to strongly degrade proteins thus dramatically reducing their IgE reactivity; characteristic protein profiles were found for specific processes such as hot and cold break. Ultra‐high‐pressure treatments were found to generally preserve the integrity of tomato proteins and this was reflected by the unaltered IgE reactivity observed. Copyright © 2007 Society of Chemical Industry     

大米蛋白酶解物的ACE抑制活性研究

为了考察大米四种蛋白经模拟体外消化后能否产生ACE抑制活性肽及其活性状况,本研究以大米为原料,Osborne法提取大米四种蛋白。体外模拟胃肠道消化过程,研究消化酶解ACE抑制活性肽产生情况及其活性大小,同时检测酶解产物的水解度和分子量分布。实验结果表明,大米四种蛋白经胃蛋白酶消化30min,酶解产物的ACE抑制活性均达到较高水平,随后经过胰蛋白酶作用,酶解产物的ACE抑制活性下降。大米清蛋白、球蛋白、醇溶蛋白和谷蛋白的4h消化产物半抑制浓度IC50值分别为1.45mg/mL、0.91mg/mL、1.19mg/mL和0.75mg/mL,分子量集中在1000u以下,是易于被人体吸收的ACE抑制活性肽。同时,未经酶解的蛋白几乎没有ACE抑制活性。结果说明大米四种蛋白的体外消化酶解物具有不同大小的ACE抑制活性,其中大米谷蛋白消化产物的ACE抑制活性最高,人体正常食用大米蛋白,经胃肠消化可以产生被人体吸收的ACE抑制活性肽。     

Separation and purification of hypocholesterolaemic peptides from whey protein and their stability under simulated gastrointestinal digestion

In this study, hypocholesterolaemic peptides were separated from whey protein trypsin hydrolysates (WPTHs) and the inhibition of cholesterol micellar solubility (ICMS) and the stability of hypocholesterolaemic peptides after exposure to simulated gastrointestinal conditions were evaluated. Whey protein trypsin hydrolysates were concentrated by ultrafiltration and then desalted through macroporous adsorption resin DA201‐C. Sephadex G‐50 gel filtration chromatography was used to separate the hypocholesterolaemic peptides from the fraction eluted with 75% ethanol. The results suggested that the fraction obtained by gel filtration with a molecular weight (MW) ranging from 1900 Da to 3100 Da exhibited the highest hypocholesterolaemic activity. This fraction was further separated by reversed‐phase high‐performance liquid chromatography into 14 fractions; the peptides with the highest activities were obtained from the fifth peak with a MW of 2454 Da and 58.77% ICMS. The hypocholesterolaemic peptides were relatively stable when exposed to simulated gastrointestinal digestion.     

海参胶的脱腥精制及体外消化特性研究

目的 以仿刺参为原料制备海参胶,通过优化最佳脱腥条件实现海参胶的精制,并探究海参胶在胃肠液中的消化特性。方法 采用单因素结合正交实验研究生姜、迷迭香、绿茶质量浓度和浸泡时间对脱腥效果的影响,以腥度值和感官评分作为标准确定最佳的脱腥优化条件。采用体外模拟消化模型,对不同消化阶段海参胶的结构变化、蛋白质溶出及蛋白质降解情况进行分析。结果 海参胶的最佳脱腥条件为:生姜、迷迭香、绿茶质量浓度分别为5.0、3.0、1.5 g/100 mL,浸泡脱腥时间为1.2 h。在模拟胃液环境中,海参胶中蛋白结构逐渐由纤维状转变为颗粒状;可溶性蛋白含量先升高后降低,游离氨基氮含量逐渐增加;分子量为100～245 kDa的高分子量蛋白丰度值相对降低, 17～35 kDa的低分子量蛋白丰度值相对增加,消化2.0 h后趋于稳定。在模拟肠液环境中,海参胶中游离氨基氮含量明显升高,低分子量蛋白继续降解为更小分子量的肽段或氨基酸。结论 本研究所得最佳脱腥精制条件可以获得具有口感独特和风味良好的海参胶制品,通过模拟胃肠液消化模型揭示了海参胶在胃部消化的相对稳定性和肠道消化的持续降解性,为海参胶制品的应用提供了理论基础和技术...     

The role of non-haem proteins in meat haemoprotein digestion

The influence of additional protein on the in vitro digestion of haem compounds was investigated. When either (59)Fe-labelled haemoglobin in blood or unlabelled purified haemoglobin were digested in vitro, the formation of low molecular weight (< 10,000), dialysable, iron degradation products was very limited (<18% of the total iron) and consisted mostly of haematin compounds. The presence of additional protein, in the form of bovine serum albumen or gelatin, greatly increased the formation of low molecular weight (<10,000) degradation products; the increase being proportional to the concentration and type of added protein. In these systems approximately two-thirds of the low molecular weight iron compounds were non-haematin in character. However, the digestion of aqueous muscle extracts resulted in the greatest formation of low molecular weight (<10,000) iron degradation products (>70% of the total iron), nearly all of which were non-haematin compounds. A hypothesis is presented explaining how haemoprotein degradation occurs in meat and related systems during normal physiological digestion.     

鸡蛋卵转铁蛋白过敏原的体外模拟消化特性

采用体外静态消化模式,分别模拟婴幼儿和成年人的胃液、小肠液以及小肠刷状缘膜酶消化鸡卵转铁蛋白,利用Tricine-SDS-PAGE和MALDI-TOF-MS对消化产物进行分析,研究鸡蛋蛋清过敏原卵转铁蛋白的消化特性.结果表明:婴幼儿和成年人的模拟胃液、小肠液以及小肠刷状缘膜酶都能够消化卵转铁蛋白.在模拟婴幼儿消化体系中...     

Comparative study of in vitro digestibility of major allergen tropomyosin and other food proteins of Chinese mitten crab (Eriocheir sinensis)

BACKGROUND: China is the largest producer and consumer of aquatic products in the world; however, many people in China suffer from allergies upon consuming crab. Stability in simulated gastric fluid is regarded as an important parameter for the estimation of food allergenicity. RESULTS: The digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Chinese mitten crab in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay systems was investigated and compared by SDS‐PAGE, western blot and inhibition ELISA. In the SGF system, proteins such as the original band of myosin heavy chain (MHC) and actin were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was easily decomposed, while TM and actin were similarly resistant to digestion. Further study by IgE immunoblotting and inhibition ELISA using sera from crab‐allergic patients indicated that allergenicity of TM was partially decreased. CONCLUSION: Chinese mitten crab major allergen TM was resistant to pepsin while relatively susceptible to trypsin and chymotrypsin digestion. Both SDS‐PAGE using purified TM and western blot using myofibrillar proteins indicated that the degradation pattern of TM by SGF and SIF was not affected by the presence of other myofibrillar proteins. Inhibition ELISA results revealed that proteinase digestion is effective in reducing the allergenicity of crab TM. Copyright © 2010 Society of Chemical Industry     

Improving the Digestibility of Lentil Flours and Protein Isolate and Characterization of Their Enzymatically Prepared Hydrolysates

Raw and cooked lentil flours and protein isolate were digested using one-step-multi-enzyme, two-step-sequential multi-enzyme or pre-hydrolyzed with one-step-single-enzyme systems. In vitro protein digestibility values ranged between 22.3 and 94.4%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed minimal loss of intact protein and more extensive digestion after actinidin and acid-protease pre-hydrolysis, respectively. Size exclusion high-performance liquid chromatography analysis confirmed greater loss of high molecular mass polypeptides after acid-protease pre-hydrolysis compared to the other enzymes. Additional digestion of pre-hydrolyzed samples using trypsin-α-chymotrypsin-peptidase resulted in further digestion and release of lower molecular mass peptides. The study demonstrates how different processing treatments and the use of enzymes (alone/in combination) influence lentil protein digestion.     

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE‐mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX‐4T‐3 and expressed in Escherichia coli BL 21. 2‐D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies. Copyright © 2011 Society of Chemical Industry     

Composition of Undigested Peptide Fragments from Gluten with Regard to Bioavailability of Essential Amino Acids

Our purpose was to isolate and characterize the high molecular weight peptides resulting from the in vitro digestion of wheat gluten in a system simulating actual in vivo conditions, and to determine to what extent essential amino acids were included in these peptides. Large peptides were separated from the smaller products by CuSephadex G-25, TLE and TLC, treated with dansyl chloride for end group determination, and redansylated to identify the remaining amino acids. Fifteen peptides, hexapeptides or larger, were isolated and were found to contain about 16.1% of the threonine, 15.2% of the methionine and 12.3% of the lysine originally present in wheat gluten.     

Molecular characterization of Api g 2, a novel allergenic member of the lipid-transfer protein 1 family from celery stalks

Scope: Celery represents a relevant cross‐reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non‐specific lipid transfer protein (nsLTP). Methods and results: MS and cDNA cloning were applied to obtain the full‐length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta‐gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α‐helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2‐specific IgE antibodies cross‐reacted with peach and mugwort pollen nsLTPs. Conclusion: Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule‐based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients.     

Immunodetection of legume proteins resistant to small intestinal digestion in weaned piglets

Experiments were conducted to investigate the biochemistry of digestion of the major storage proteins from soya bean, pea, faba bean, blue lupin, and chickpea seeds in the ileum of piglets. Hyperimmune plasmas against the crude protein extracts and the purified 11S and 7S globulin fractions of each legume seed and an anti‐pea albumin PA2 and lectin antibody were used. They served to probe immunoblots of feed protein extracts and ileal digesta samples. Globally, the recognition by plasmas of intact or partially digested proteins in ileal digesta was rather faint, in agreement with the fairly high in vivo digestibility data obtained with these legume seed proteins. Nevertheless, immunoreactive polypeptides found in digesta of piglets fed pea, faba bean and chickpea belonged mainly to proteins of the 7S family, and to other proteins including low‐molecular weight components such as PA2 albumin and lectin in the case of pea. In piglets fed lupin, nearly intact polypeptides from the 11S family were detected. To conclude, the present immunochemical study conducted on ileal digesta of piglets revealed a few dietary legume proteins of the vicilin and albumin families. Legumin proteins were demonstrated unequivocally in the case of lupin and white chickpea. Copyright © 2003 Society of Chemical Industry     

Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (< 14.4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin-proteinase K complex and its sequence determined to be Val-Ala-Gln-Gly-Gly-Ala-Ala-Gly-Leu-Ala. Circular dichroism studies indicated a high alpha-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.