山羊乳与牛乳配方乳粉的致敏性比较

山羊乳是一类营养丰富且易于消化的乳品,因其营养成分与牛乳相近而受到广泛关注。本研究通过蛋白质潜在致敏性树状评估策略（生物信息学、消化稳定性、血清学研究、动物模型）对山羊乳配方乳粉的致敏性进行评价,并研究山羊乳配方乳粉与牛乳过敏患者血清中免疫球蛋白（immunoglobulin,Ig）E之间存在的交叉反应性。结果表明,山羊乳配方乳粉的167 种蛋白质中,β-乳球蛋白和α-乳清蛋白含量显著低于牛乳配方乳粉（P＜0.05）,山羊乳配方乳粉在模拟胃液中更易消化,且与过敏患者血清的结合能力弱于牛乳配方乳粉,动物模型结果表明山羊乳配方乳粉致敏小鼠体温下降幅度、临床过敏症状评分、血清中特异性IgE和IgG1抗体水平、血浆中组胺水平、血管渗透性及组织炎症程度等均显著低于牛乳配方乳粉组致敏小鼠（P＜0.05）,交叉反应性结果表明山羊乳配方乳粉能与牛乳过敏患者血清结合,但结合能力明显低于牛乳配方乳粉。结论：与牛乳配方乳粉相比,山羊乳配方乳粉的致敏性较弱,可作为牛乳的替代品,降低牛乳过敏性疾病的发病风险。     

转基因牛的重组人α-乳清白蛋白的离体和体外消化稳定性研究

目的 通过测定转人α-乳清白蛋白基因的克隆牛所表达的重组人α-乳清白蛋白在模拟胃肠液及小型猪胃肠液中的消化时间,并与人α-清白蛋白标准品在上述消化液中的消化时间相比较,判定该重组蛋白的消化稳定性,为其致敏性评价提供基础资料.方法 根据<转基因生物及其产品食用安全检测--模拟胃肠液外源蛋白质消化稳定性试验方法>消化人α-...     

利用血清筛选和模拟胃肠液消化稳定性试验评价植物源重组人血清白蛋白潜在致敏性

目的 利用血清筛选试验和模拟胃肠液消化稳定性试验,探讨植物源重组人血清白蛋白(OsrHSA)是否具有潜在致敏性。方法 选择对虾、小麦、屋尘螨、鸡蛋、牛奶过敏,特异性IgE抗体浓度大于3.5 kUA/L的患者血清75份和健康人血清4份,利用酶联免疫吸附试验探讨OsrHSA能否与过敏血清中的过敏原特异性IgE抗体结合;按照国家标准《转基因生物及其产品食用安全检测模拟胃肠液外源蛋白质消化稳定性试验方法》进行OsrHSA的模拟胃肠液消化稳定性试验,评估OsrHSA的消化稳定性。结果 有3份对牛奶过敏的血清与OsrHSA发生免疫交叉反应,该蛋白与牛奶过敏原可能存在抗原交叉性;OsrHSA在模拟胃肠液中15 s内即被消化完全,表明该蛋白极易被消化。结论 OsrHSA的潜在致敏性较低,对牛奶过敏的人群需要慎重使用。     

超声波辅助糖基化对 β-乳球蛋白消化过程中致敏性的影响

采用间接竞争ELISA、扫描电镜和动态光散射等技术,研究超声波结合乳糖糖基化修饰对 β-乳球蛋白(β-Lg)在体外模拟消化过程中致敏性和结构变化的影响.结果表明:被修饰的β-Lg在消化过程中致敏性显著降低,胃肠消化的水解度也降低;被修饰的的β-Lg经胃消化形成更大的颗粒结构,在肠消化中发生聚集;消化后其荧光强度先升高后...     

重组人α-乳清白蛋白潜在致敏性的生物信息学预测

目的 通过生物信息学分析预测牛乳腺生物反应器表达的重组人α-乳清白蛋白(rhLA)的致敏性.方法 使用在线致敏原数据库(The Allergen Online Database)、致敏蛋白结构数据库(Structural Database of Allergenic Proteins)和食品安全致敏原数据库(Allergen Database for Food Safety),分析目标蛋白与已知致敏原的序列或结构相似性.结果 rhLA致敏性的生物信息学分析结果显示,rhLA与已知致敏原牛α-乳清白蛋白、牛乳糖合酶B蛋白(致敏原Bos d 4)、鸡溶菌酶C(1,4-β-N乙酰胞壁质酶C、致敏原Gal d Ⅳ、致敏原Gal d 4)存在较高的序列或结构同源性.结论 rhLA具有潜在的致敏可能性.     

蟹类主要过敏原的模拟肠胃液消化及其对过敏原活性的影响

目的:通过模拟肠胃液消化试验,分析锯缘青蟹肌肉中过敏原(原肌球蛋白)的消化特性以及消化对过敏蛋白致敏性的影响.方法:采用美国药典提供的模拟胃、肠液配方,测定青蟹原肌球蛋白和肌原纤维蛋白在体外模拟肠胃环境中的消化稳定性.以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹法(Western-blot)进行分析.结果:在模拟胃液反应中,非过敏原蛋白尤其是肌球蛋白重链和肌动蛋白等被胃蛋白酶快速降解,而原肌球蛋白在60 min时仍未被完全分解.在模拟肠液反应中,相对于致敏蛋白,肌球蛋白重链、肌动蛋白等非过敏蛋白在较短的时间内被分解,原肌球蛋白尽管120 min后几乎被完全分解,但会产生一些较稳定的蛋白降解片段.以免疫印迹法检测过敏患者血清,结果分子质量为34 kDa的原肌球蛋白降解产物仍具有过敏原性.结论:蟹类过敏蛋白相对于非过敏蛋白具有较高的消化稳定性,而且其高分子质量降解产物仍可能引起过敏反应.     

模拟消化方法在食物过敏原潜在致敏性评价中的应用进展

食物过敏已成为全球范围内日益严重的食品安全问题。对食物过敏原的潜在致敏性进行评价有助于更好地了解食物过敏原自身特性及其对过敏人群免疫系统的影响。模拟消化实验是食物过敏原潜在致敏性评价的有力手段。食物过敏原蛋白的消化稳定性为其潜在致敏性评价结果提供了重要参考依据,但并非所有食物过敏原均具有较强的消化稳定性。食物过敏原的潜在致敏性一方面取决于过敏原蛋白在通过消化道时的消化稳定性,另一方面取决于消化产物中具有免疫刺激能力的过敏原表位的丰度。不同模拟消化方法的应用对食物过敏原的消化结果及潜在致敏性评价结果的真实性和可比性具有重要影响。本文系统地介绍了体内及体外模拟消化方法的优缺点及在食物过敏原潜在致敏性评价中的应用研究进展,并综述了食物过敏原消化稳定性与其潜在致敏性评价结果的关系。以期对食物过敏原致敏性评价体系的进一步完善和发展有所帮助。     

BALB/c小鼠动物模型评价食物过敏性的可行性研究

目的:动物模型对于模拟人体食物过敏的可能性以及用来评价食品致敏性的可行性一直存在争议。本研究中使用BALB/c小鼠研究了其作为食物过敏动物模型的可行性,并对几种食物中蛋白的致敏性进行评价。方法:选择大豆球蛋白、卵清白蛋白、马铃薯碱性磷酸酶作为受试物,对各蛋白进行BALB/c小鼠模型研究,模拟胃液消化试验和生物信息学分析。取3~4周龄雌性BALB/c小鼠,经口给予蛋白诱发致敏。大刺激后,测定动物血浆中组胺水平,血清中特异性Ig E、Ig G1水平,m MCP-1水平和肠道渗透性,并对主要脏器进行病理组织学观察。结果:给予BALB/c小鼠一次经口致敏各蛋白,各组间特异性抗体Ig E、Ig G1水平、m MCP-1水平、组胺水平以及白蛋白水平均未表现出明显差异;而给BALB/c小鼠多次接触过敏原时,特异性抗体Ig E、Ig G1水平、组胺以及白蛋白水平都有显著性升高,产生明显的Th2型免疫反应,且BALB/c小鼠能区分这些蛋白的致敏性,即大豆球蛋白卵清白蛋白马铃薯碱性磷酸酶。模拟胃液消化试验也验证了动物试验对受试蛋白致敏性的分析结果。通过生物信息学对抗原指数分析,同样发现大豆球蛋白致敏性显著高于卵清白蛋白和马铃薯碱性磷酸酶。结论:BALB/c小鼠用于研究食品过敏原中的大豆球蛋白、卵清白蛋白、马铃薯碱性磷酸酶的致敏性是可行的,能够区分这些食物蛋白的致敏性。     

体外消化模型评估食物过敏原致敏性研究进展

食物过敏受到了人们的广泛关注,检测和评价食物过敏原的潜在致敏性日益受到重视。体外胃肠道消化实验是食物过敏原潜在致敏性评价体系的重要环节之一。该文系统介绍体外胃肠道消化模型的种类及其应用,并综述体外消化模型与体内消化模型相比的优缺点,最后对食物过敏原消化稳定性与潜在致敏性的关系进行探讨,以期为食物过敏原潜在致敏性评价研究中体外模拟消化方法的应用及消化结果分析提供参考。     

乳铁蛋白分离纯化研究进展

乳铁蛋白是一种非血红素转铁蛋白,与血清转铁蛋白、卵转铁蛋白和黑素转铁蛋白共同组成转铁蛋白家族。乳铁蛋白是一种多功能性的糖蛋白,具有很多的活性,被广泛应用于保健食品、配方奶粉、医药等领域中。归纳传统的与新开发的乳铁蛋白的回收方法,比较每种方法在提取回收乳铁蛋白中的优势与不足,对今后开发出更佳具有优势的乳铁蛋白的回收方法提供一些思路。     

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.     

Comparative study of in vitro digestibility of major allergen,tropomyosin and other proteins between Grass prawn (Penaeus monodon) and Pacific white shrimp (Litopenaeus vannamei)

BACKGROUND: Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity. In the present study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Grass prawn and Pacific white shrimp in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and comparatively studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), western blotting, and inhibition enzyme‐linked immunosorbent assay (ELISA). RESULTS: In the SGF system, proteins such as actin and myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was also easily decomposed, while TM and actin were resistant to digestion. Western blotting using a specific polyclonal antibody against TM indicated that the degradation pattern of shrimp TM by SGF and SIF was almost unaffected by the presence of other myofibrillar proteins. Further study by IgE immunoblotting and inhibition ELISA using sera from crustacean‐allergic patients indicated that IgE binding of TM was decreased. CONCLUSION: Proteinase digestion is effective in reducing IgE binding of shrimp TM. It is also of interest to notice that Pacific white shrimp TM had higher digestion stability than Grass prawn TM. However, Pacific white shrimp TM revealed enhanced IgE binding over that of TM from Grass prawn and thus it is possibly more allergenic. Copyright © 2010 Society of Chemical Industry     

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     

中国食物过敏原数据库的建立与应用

目的氨基酸序列相似性等生物信息学分析方法是转基因食品致敏性评价的重要环节,为了简化转基因食品致敏性生物信息学分析的流程,我们建立了中国食物过敏原数据库。方法将多个数据库中关于过敏原的信息进行了整合并进行本地存储。通过web语言PHP、web服务器Apache和MySQL数据库管理系统进行搭建数据库。结果建成了中国食物过敏原数据库,收集到1498条已知过敏原的记录,数据库发布在hap://175.102.8.19:8001/site/index。结论该数据库可以为科研人员提供免费的、快速的、一站式的转基因食品致敏性分析信息服务。     

Mitigation of peanut allergenic reactivity by combined processing: Pressured heating and enzymatic hydrolysis

Among food allergens, peanut is one of the most critical. This study evaluates peanut allergenic features after the combination of heat, pressure, and enzymatic digestion under sonication, by immunodetection using serum IgE of sensitized patients and mass-spectroscopy. In the studied population, there was a predominance of patients with sensitization to Ara h 9 (nsLTP) followed by sensitization to seed storage proteins (Sprot, Ara h 1, 2, 3, and 6). The Sprot sensitized patients showed higher reactivity. The enzyme E5 was efficient for inducing protein fragmentation and allergenic reactivity reduction when it was used combined with pressured heating treatments such as autoclave and Controlled Instantaneous Depressurization (DIC). Only a few Ara h 1 and Ara h 3 peptides were identified after enzymatic digestion of DIC peanut samples. The combination of pressured heating treatments and enzymatic hydrolysis was the most efficient method to strongly mitigate or even eliminate the allergenic potential of peanut. Our findings set a possibility for a group of patients in which their allergy could be treated with a processed less-allergenic peanut and consequently less risky, more easy and quicker desensitization treatment.Industrial relevanceThe findings identify innovative thermal, pressure and enzymatic processing conditions highly effective to mitigate or even abolish the allergenic potency of peanut, which may be relevant for consumers, clinicians, regulatory agencies and the food industry. The applications of processed peanut with reduced IgE binding potency for tolerance induction might be a convenient strategy.     

食品过敏原检测与评价技术研究进展

过敏原生物活性包括过敏原性即引起致敏个体发生过敏症的活性和致敏原性即引起人群致敏的危险性两个方面。随着转基因作物及相应食品的大量出现，评价和检测食品过敏原生物活性日益受到重视。迄今，食品过敏原性的主要检测方法有：皮肤试验、双盲安慰剂对照激发试验、血清IgE检测和组胺释放试验等；对食品致敏原性还没有建立可靠的评价和监测技术，FAO/WHO采纳的分级评价策略包括血清学测定、过敏原分子结构和序列同源性比较及胃肠液消化稳定性评价等。本文对食品过敏原生物学活性的评价与检测技术研究进展进行综述。     

Food processing increases casein resistance to simulated infant digestion

The objective of this study was to determine whether processing could modify the resistance of casein (CN) to digestion in infants. A range of different dairy matrices was manufactured from raw milk in a pilot plant and subjected to in vitro digestion using an infant gut model. Digestion products were identified using MS and immunochemical techniques. Results obtained showed that CNs were able to resist digestion, particularly κ‐ and αs₂‐CN. Resistant areas were identified and corresponded to fragments hydrophobic at pH 3.0 (gastric conditions) and/or carrying post‐translational modifications (phosphorylation and glycosylation). Milk processing led to differences in peptide patterns and heat treatment of milk tended to increase the number of peptides found in digested samples. This highlights the likely impact of milk processing on the allergenic potential of CNs.     

Identification of the Main Allergenic Proteins in High Hydrostatic Pressure Pineapple Juice and Assessing the Influence of Pressure on their Allergenicity

The objective of this work was to identify the main allergenic proteins and to assess the influence of high hydrostatic pressure on the potential allergenicity of high hydrostatic pressure pineapple juice. Fresh pineapple juice was treated with different pressures and the allergenic proteins were extracted and analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis, Western bloting and liquid chromatography-tandem mass spectrometry. The main allergenic proteins were detected as 24-kDa and 14-kDa bands by serum of pineapple-allergic patients. liquid chromatography-tandem mass spectrometry analysis of these bands showed that they actually consisted of a mixture of several isotypes of cysteine proteinases. Among these, bromelain (Ananas comosus) is a known allergen named Ana c 2 and it is highly homologous to other cysteine proteinases. Enzyme-linked immunosorbent assay assay of high hydrostatic pressure pineapple juice carried out at ambient temperature (18–22°C) showed that the higher the pressure, the higher the reduction in allergenicity, yielding a maximum reduction of about 20% under 500 MPa. However, in the case of 400 MPa and 50°C, the reduction rate of allergenicity was higher, up to 50% compared to that of the sample at 20°C without high hydrostatic pressure treatment. Taken together, the results suggested that high-pressure treatment could alter the potential allergenicity of pineapple juice allergens and identified Ana c 2 and a few other related cysteine proteinases as the main allergens.     

Allergenicity of roasted peanuts treated with a non-human digestive protease

Peanut allergy is a severe and lifelong type of food allergy triggered by allergenic proteins and peptides in peanuts. This study investigated the effects of ultrasound-assisted alcalase treatment on the concentrations of major allergenic proteins (Ara h 1 and Ara h 2) in roasted peanut kernels and the allergenicity of treated peanut extracts. Peanut kernels were sonicated for 1 h in buffer solution, incubated with different amount of alcalase for various time, then vacuum dried. The variations of Ara h 1 and Ara h 2 contents in soluble and insoluble portions of peanuts treatments were evaluated by sandwich ELISA and SDS-PAGE, respectively. The in vitro IgE-binding capacity of treated peanut extracts was determined by a competitive inhibition ELISA using pooled plasma of 10 peanut allergic patients. Samples with lower in vitro IgE-binding were used for human skin prick tests (SPTs) in peanut allergic individuals. Results indicate that alcalase digestion of sonicated peanuts significantly increased protein solubility while decreasing Ara h 1 and Ara h 2 concentrations in both soluble and insoluble portions of peanuts relative to untreated peanuts. The maximum reductions of Ara h 1 and Ara h 2 levels were obtained following 3 hour digestion with alcalase at concentrations of 4.54 and 6.05 U/100 g. Samples obtained under these conditions showed the lowest in vitro IgE-binding and caused the least allergic response in human SPTs. The current study suggests that the allergenic potential of peanuts could be reduced by postharvest processing such as ultrasound-assisted enzymatic treatment of peanuts kernels.