酪蛋白糖巨肽对小鼠腹腔吞噬细胞及肠黏膜免疫细胞的影响

目的：由于酪蛋白糖巨肽（casein glycomacropeptide,CGMP）具有许多生理活性功能和独特营养特性,研究CGMP对小鼠腹腔免疫细胞以及肠黏膜屏障的影响可为其应用于保健食品和医药品提供科学依据。方法：以96 只健康昆明雌性小鼠为实验动物,利用流式细胞仪、HE染色和免疫荧光标记技术,分别检测分析了灌胃不同剂量的CGMP（30 μg/d和120 μg/d）对实验小鼠腹腔吞噬细胞吞噬活性以及肠上皮内淋巴细胞（intraepithellallymphocytes,IEL）和固有层IgA+浆细胞数量的影响,探讨CGMP改善小鼠腹腔吞噬细胞和肠黏膜屏障功能的作用。结果：CGMP能够显著增加小鼠腹腔吞噬细胞的吞噬能力,灌胃时间越长,作用效果越明显;其中灌胃CGMP的剂量为120 μg/d的作用效果优于30 μg/d剂量组。且与对照组相比,灌胃CGMP组小鼠十二指肠上皮内淋巴细胞数和固有层IgA+浆细胞数量显著增加（P ＜ 0.01）。结论：CGMP可以增强小鼠腹腔吞噬细胞吞噬功能,诱导肠道黏膜免疫应答,增强肠黏膜免疫屏障功能,有望开发为抑制肠道炎症的功能性食品添加剂。     

分离β-伴大豆球蛋白和大豆球蛋白工艺研究

采用大容量(250×4mL)低速(5300×g)离心机分离β-伴大豆球蛋白和大豆球蛋白,进行碱溶条件、酸沉条件等条件的研究。结果表明:提取蛋白的碱性溶液(pH8.5的Tris-HCl溶液)离心效果较佳,沉淀大豆球蛋白pH6.4为佳,沉淀β-伴大豆球蛋白pH4.8为佳,用pH5.0沉淀杂蛋白离心分离效果较好;每一个待离心的浊液,在离心之前4℃冷藏2h以上,对离心效果的提高十分有效;采用最佳条件,从300g脱脂豆片分离大豆球蛋白和β-伴大豆球蛋白,收率分别为6.8%和2.2%,用SDS-PAGE电泳实验方法测定大豆球蛋白和β-伴大豆球蛋白样品纯度分别为89.1%和78.9%。因此该低速离心法较适合数十克级别的β-伴大豆球蛋白和大豆球蛋白的分离。     

β-伴大豆球蛋白的水解研究

采用Nagano法从豆粕中分离β-伴大豆球蛋白并酶解制备水解肽,以单因素试验和正交试验确定酶解最佳条件,通过高效液相法分析β-伴大豆球蛋白水解肽的分子量分布,比较并检测了β-伴大豆球蛋白水解肽和大豆分离蛋白水解肽的体外抗氧化效果。结果显示:在20g/L的底物浓度下的最佳条件为酶和底物比10000U/g,温度55℃,pH7.5,水解时间4h,水解度为72.7%,明显高于酶解大豆分离蛋白51.4%的水解度,且水解时间更短。β-伴大豆球蛋白水解肽主要为130～1000u的短肽,占肽总量的86.3%,均一性极高。β-伴大豆球蛋白水解肽对O2-.和.OH均有清除作用,清除.OH的能力明显高于大豆分离蛋白水解肽,即β-伴大豆球蛋白的水解肽对大豆肽清除.OH的作用贡献更大。     

β-伴大豆球蛋白糖基化产物的致敏性研究

为探究食品热加工过程中糖基化产物对蛋白质致敏性的影响,该文模拟3种不同加工条件,制备β-伴大豆球蛋白的糖基化产物,利用β-伴大豆球蛋白致敏模型,对糖基化产物的致敏性进行评价,并探讨β-伴大豆球蛋白结构变化与致敏性的关系。检测小鼠过敏症状评分、体重、免疫球蛋白E（immunoglobulin E,IgE）、组胺、类胰蛋白酶以及接枝度、内源荧光性和二级结构。结果表明,与低温糖基化产物和蒸煮糖基化产物相比,高压灭菌糖基化产物的结构变化最显著,引起小鼠致敏反应最弱。     

枸杞多糖对小鼠肠道上皮内淋巴细胞和杯状细胞数量、分布及对IL-2水平影响

为了研究枸杞多糖(LBP)对肠黏膜屏障结构的影响,给10只C57BL/J健康小鼠连续灌服枸杞多糖7d后,显微镜观察小肠上皮内淋巴细胞(IEL)、杯状细胞(GC)数量和分布变化及测定肠黏膜白细胞介素-2(interleukin-2,IL-2)水平。结果表明,应用枸杞多糖小鼠的肠黏膜IEL数量和GC的数量明显增加,且有向肠内层移动趋势,同时肠黏膜IL-2水平升高。证实枸杞多糖在一定程度上可促进小鼠小肠黏膜屏障结构趋于完整并提高其免疫功能。     

大豆多糖对环磷酰胺抗肿瘤作用的减毒增效机制研究

研究大豆多糖能否减轻环磷酰胺对机体免疫系统和造血系统的影响。以昆明种小鼠为研究对象,腋下接种S180肉瘤,腹腔注射环磷酰胺25mg/kg·d以及大豆多糖50、100、200mg/kg·d。测定S180荷瘤小鼠外周血中白细胞数量,ELISA法检测细胞因子TNF-α的变化,流式细胞仪检测S180荷瘤小鼠T淋巴细胞亚群CD+4/CD+8变化。结果显示:与单独应用环磷酰胺相比,大豆多糖可以提高荷瘤小鼠的外周血中白细胞数量;提高荷瘤小鼠血清中细胞因子TNF-α水平。随大豆多糖给药剂量的升高,CD+4细胞比例变化不明显,CD+8细胞比例降低,CD+4/CD+8升高。故大豆多糖能够通过调节荷瘤小鼠免疫功能而增强环磷酰胺的抗肿瘤作用,并减轻其毒副作用。     

透明质酸-大豆β-伴球蛋白涂膜对微冻鲤鱼的保鲜效果

目的 研究不同比例透明质酸和大豆β-伴球蛋白复合膜处理对鲤鱼肉微冻贮藏品质的影响。方法 用浓度为0.9%的 HA分别和1%、2%、3%大豆β-伴球蛋白配制三种涂膜剂,涂膜分割鲤鱼肉后进行-3℃微冻贮藏,测定鲤鱼肉持水力、电导率、pH、色度,挥发性盐基氮、硫代巴比妥酸值、TCA可溶性肽以及肌原纤维蛋白、表面疏水性、羰基、巯基和Ca_²⁺-ATPase活性的变化。结果 未涂膜鲤鱼肉在微冻贮藏14 d后品质显著下降,在微冻贮藏28 d内,与未涂膜鲤鱼肉相比,HA-大豆β-伴球蛋白复合膜可有效抑制微冻鲤鱼肉持水力、肌原纤维蛋白、巯基和Ca_²⁺-ATPase活性的下降(p＜0.05),延缓TVB-N、TBARS、TCA可溶性肽、电导率和羰基的增长速率(p＜0.05)。0.9%HA-2%大豆β-伴球蛋白复合膜保鲜28 d时,与未涂膜鲤鱼肉理化指标相比,其电导率低20.2%, TVB-N低48.9%,TBARS低62.6% ,TCA可溶性肽低20.7%;肌原纤维蛋白含量和持水力分别高40.6%和27.9%。结论 0.9%HA-2%大豆β-伴球蛋白复合膜保鲜效果最好,该涂膜剂可有效延缓微冻鲤鱼肉的劣变,延长货架期。     

加热联合酶解大豆7S蛋白的分子结构及抗原性研究

选用不同加热预处理条件、不同蛋白酶处理大豆7S蛋白,SDS-PAGE考察酶解后大豆7S蛋白的降解情况,间接竞争ELISA法考察酶解物的抗原性。结果表明:加热预处理联合酶解可显著增大β-伴大豆球蛋白的降解程度,β亚基消化程度得到极大提升,α亚基、α'亚基几乎完全被降解,在相同酶解时间下,胰蛋白酶的酶解速度快于胃蛋白酶;加热预处理联合酶解大幅降低了β-伴大豆球蛋白的抗原性,胰蛋白酶的效果优于胃蛋白酶,经80℃加热10 min预处理再经胰蛋白酶处理,大豆7S蛋白的抗原性降低了79.99%,而胃蛋白酶最优条件下最多仅降低50.4%; SDSPAGE结果表明,胃蛋白酶、胰蛋白酶酶解物中出现了抗消化肽段,经过质谱鉴定,均来自于β-伴大豆球蛋白的α亚基。     

副干酪乳杆菌L9对过敏小鼠淋巴细胞Th1/Th2平衡的调节作用

目的:研究副干酪乳杆菌L9对牛乳β-乳球蛋白(BLG)过敏小鼠淋巴细胞Th1/Th2平衡的影响,探讨L9缓解机体过敏反应的机制。方法 :构建BLG过敏小鼠模型,通过ELISA方法和流式细胞术分析L9对过敏小鼠原代淋巴细胞和骨髓源树突状细胞(BM-DCs)细胞因子分泌及调节性T细胞(Foxp3+Treg)数量的影响。结果:不同剂量的活/热致死L9均可显著提高过敏小鼠淋巴细胞上清中IFN-γ水平,显著降低IL-4含量,减少BLG特异性抗体的产生(P0.05);显著促进BM-DCs细胞上清中调节性细胞因子IL-10、TGF-β的分泌(P0.05),提高过敏小鼠CD4+T淋巴细胞中Foxp3+Treg细胞比例,并且活菌与热致死菌的调节效果相似。结论 :L9能够调节过敏小鼠淋巴细胞的Thl/Th2失衡,这可能与树突状细胞和Foxp3+Treg介导的免疫抑制有关。     

抗疲劳酸乳的研制

以新鲜牛奶和黑米为原料,添加大豆β-伴球蛋白肽,通过发酵研制出具有抗疲劳功效的保健型酸乳.发酵前添加大豆β-伴球蛋白肽,最佳添加量为1％;利用响应面设计,优化出发酵条件:磨浆比为1∶7,黑米汁和鲜牛乳配比为40∶63,接种量为7％.该产品为浅紫色凝乳,表面光滑,质地细腻均匀,具有黑米香味和清香味,酸甜爽口.     

Influence of NaCl concentration and high pressure treatment on thermal denaturation of soybean proteins

In the present work the effect of high pressure (HP) treatment in the presence of NaCl on the thermal behavior of soybean proteins was analyzed by differential scanning calorimetry. The thermograms obtained have shown that NaCl addition increased the thermal stability – increase in temperatures of denaturation (Td) – of both glycinin and β-conglycinin. HP treatments increased thermal stability of glycinin, but decreased that of β-conglycinin. High NaCl concentrations decreased (in glycinin) or inverted (in β-conglycinin) the effects of HP on thermal stability. Cooperativity of denaturation of glycinin was enhanced by NaCl and HP. Cooperativity of denaturation of β-conglycinin was enhanced by HP and also by NaCl at 0.2 mol/L but decreased with the combination of treatments. Salt addition increased the enthalpy, ΔH, of denaturation of glycinin and β-conglycinin, being this effect stronger on glycinin. HP treatment provoked the denaturation of both protein fractions. The presence of NaCl protected glycinin against HP-denaturation at any assayed salt concentration and pressure level, while β-conglycinin was only protected at 200 and 400 MPa, but was more denaturated at 600 MPa in the presence of 0.6 mol/L of NaCl.

Industrial relevance

The knowledge provided by this work may be useful in the handling of high pressure-treated food with high NaCl content (e.g. meat emulsions, smallgoods) where soybean proteins are used as additives, in order to choose high pressure values to achieve their denaturation or predict the effects of ulterior thermal treatments. Thus, this knowledge may be useful to increase the use of high pressure in food industry.     

微生物发酵对豆粕抗原性的影响

探讨了微生物发酵对豆粕抗原性的影响。选用植物乳杆菌、干酪乳杆菌、枯草芽孢杆菌、地衣芽孢杆菌和米曲霉这5种菌株,在液态和固态条件下分别发酵豆粕12 h,对发酵产物进行抗原性测定。结果表明:豆粕经这5种菌株发酵后,粗蛋白含量均有所提高,其中枯草芽孢杆菌在固态发酵时降解豆粕抗原蛋白和降低豆粕抗原性的效果优于其它菌株,此时,豆粕蛋白水解度为4.89%,必需氨基酸含量为193.51mg/g。SDS-PAGE显示发酵豆粕中β-伴大豆球蛋白的α’和α亚基消失,β亚基条带和大豆球蛋白的酸性亚基条带密度减弱,同时大豆球蛋白与β-伴大豆球蛋白的抗原性降低率分别为20.62%和50.12%。     

酪蛋白糖巨肽对小鼠肠道免疫系统的影响

研究酪蛋白糖巨肽(CGMP)对正常小鼠肠相关淋巴组织的免疫应答反应的影响,对阐述CGMP 的生物学功能具有重要意义。本实验将BALB/c 雌性小鼠28 只随机分为4 组,每组7 只,分别为0.9% 生理盐水对照组(0.2mL/d),低、中、高3 种剂量组(30、120、300μg/d)。连续灌胃16d 后,取脾脏、PP 结(peyer s patches)和肠系膜淋巴结(mesenteric lymph node,MLN),制备细胞悬液,进行三色荧光标记后,用流式细胞仪检测CD3+、CD3+CD4+ 和CD3+CD8+T 淋巴细胞亚群的变化。结果表明：CGMP 各剂量组均能够促进脾脏和PP 结中CD3+ 和CD3+CD4+ 细胞的显著增加(P ＜ 0.05),高剂量能引起脾脏CD3+CD8+ 淋巴细胞的显著增多(P ＜ 0.05),低剂量能引起脾脏CD3+CD4+/ CD3+CD8+ 的显著增加(P ＜ 0.05);低剂量CGMP 能使MLN 中CD3+CD8+ 淋巴细胞显著增减少(P ＜ 0.01)。研究证实长期灌胃CGMP 会诱导肠黏膜产生获得性的免疫应答,并且不会引起机体产生口服耐受。     

In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit

Glycinin, basic subunit and β-conglycinin were isolated from soybean protein isolate and tested for their antimicrobial action against pathogenic and spoilage bacteria as compared to penicillin. The three fractions exhibited antibacterial activities equivalent to or higher than penicillin in the next order; basic subunit>glycinin>β-conglycinin with MIC of 50, 100 and 1000 μg/mL, respectively. The IC(50%) values of the basic subunit, glycinin and β-conglycinin against Listeria\monocytogenes were 15, 16 and 695 μg/mL, against Bacillussubtilis were 17, 20, and 612 μg/mL, and against S. Enteritidis were 18, 21 and 526 μg/mL, respectively. Transmission electron microscopy images of L. monocytogenes and S. Enteritidis exhibited bigger sizes and separation of cell wall from cell membrane when treated with glycinin or basic subunit. Scanning electron microscopy of B. subtilis indicated signs of irregular wrinkled outer surface, fragmentation, adhesion and aggregation of damaged cells or cellular debris when treated with glycinin or the basic subunits but not with penicillin. All tested substances particularly the basic subunit showed increased concentration-dependent cell permeation assessed by crystal violet uptake. The antimicrobial action of glycinin and basic subunit was swifter than that of penicillin. The cell killing efficiency was in the following descending order; basic subunit>glycinin>penicillin>β-conglycinin and the susceptibility of the bacteria to the antimicrobial agents was in the next order: L. monocytogenes>B. Subtilis>S. Enteritidis. Adding glycinin and the basic subunit to pasteurized milk inoculated with the three bacteria; L. monocytogenes, B. Subtilis and S. Enteritidis (ca. 5 log CFU/mL) could inhibit their propagation after 16-20 days storage at 4 °C by 2.42-2.98, 4.25-4.77 and 2.57-3.01 log and by 3.22-3.78, 5.65-6.27 and 3.35-3.72 log CFU/mL, respectively.     

Gelation of soybean proteins induced by sequential high-pressure and thermal treatments

The effect of high-pressure treatment on structural and rheological properties of soybean protein dispersions was studied. A sequential high-pressure/thermal treatment was also analyzed. Dissimilar effects on soy protein isolate (SPI) and the enriched soybean protein fractions: β-conglycinin (βCEF) and glycinin (GEF) were observed. High pressure (600 MPa) promoted βCEF gelation, but did not modify the rheological properties of GEF in spite of its complete denaturation. Pressure treatment also induced the establishment of hydrophobic interactions and disulfide bonds that allowed the formation of soluble high molecular mass aggregates from the different polypeptides of both β-conglycinin and glycinin. Protein strands formation was detected in matrix microstructure of HP-treated SPI and βCEF dispersions in accordance with their rheological behavior of weak gels. In the case of GEF modifications induced by HP in the microstructure (apparition of large granules) were not accompanied by rheological changes.     

大豆球蛋白自组装聚集及凝胶特性

研究了在低pH值、低离子强度下,分别加热诱导不同浓度11S(大豆球蛋白)和7S(大豆伴球蛋白)自组装纤维化聚集体的形成。通过平均粒径和Thioflavin T(硫磺素T)荧光光谱,对自组装纤维化聚集体的性质进行表征,并对其热致凝胶的流变学,硬度和微结构特性进行考察。结果表明:在低pH值、低离子强度的诱导条件下,蛋白浓度对自组装聚集的形成起着关键作用,随着诱导浓度的增大,蛋白的纤维化聚集越明显,7S比11S更容易形成纤维化聚集。在酸性环境下,大豆球蛋白的纤维化聚集程度越高,越有利于热致凝胶网络结构的形成。在相同的预处理条件下,11S自组装凝胶硬度强于7S。扫描电镜结果显示7S自组装凝胶的网络结构较11S致密,但有序性较11S低。     

乳源酪蛋白糖巨肽对小鼠外周血T淋巴细胞亚群的影响

目的：酪蛋白糖巨肽（caseino-glycomacropeptide,CGMP）是κ-酪蛋白经凝乳酶和胃蛋白酶酶解后形成的含有64 个氨基酸的不同种类肽段,具有多种生物学活性和独特的营养特性,探讨CGMP对小鼠外周血T淋巴细胞亚群的影响。方法：以健康小鼠为实验动物,利用流式细胞仪分别检测长期和短期灌胃不同剂量的CGMP（30、60、120 μg/d）后,小鼠外周血淋巴细胞亚群的变化。结果：乳源CGMP干预后的结果表明,在短期灌胃实验中,灌胃不同剂量后CD3+淋巴细胞数比例均显著降低,12 h后恢复灌胃前水平;CD3+CD4+和CD3+CD8+淋巴细胞比例在灌胃30 μg和120 μg CGMP 4 h后均显著降低,而60 μg组则无明显变化;长期灌胃实验中,在第2天和第4天,外周血中CD3+淋巴细胞和CD3+CD4+比例与对照组相比显著升高（P＜0.05）;120 μg/d剂量组的小鼠外周血中CD3+CD8+淋巴细胞比例与对照组相比显著升高（P＜0.05）,而30 μg/d剂量组在2、4、6、8 d与对照组相比无显著变化（P＞0.05）;在第2天,30 μg/d和120 μg/d剂量组外周血中CD3+CD4+/ CD3+CD8+比例与对照组相比显著升高（P＜0.05）。结论：乳源CGMP能够引起正常小鼠的外周血淋巴细胞亚群发生变化,调节机体T淋巴细胞亚群的平衡,由此提示CGMP可作为功能性食品对外周血T淋巴细胞亚群的失衡有缓解作用。     

Temperature Effect on Binding of Volatile Flavor Compounds to Soy Protein in Aqueous Model Systems

The thermodynamics of binding was determined for soybean glycinin and β-conglycinin and flavor ligands butanal, pentanal, hexanal, octanal, 2- and 3-hexanone, 2- and 5-nonanone, hexanol, and hexane. Hexane had affinity for these proteins only at 5^oC. Affinities of binding for all flavor ligands were greater for glycinin than β-conglycinin. Affinity for aldehydes increased with increasing chain length for glycinin, but remained constant for β-conglycinin. ΔG^o, ΔH^o, and ΔS^o for binding were determined. ΔG^o was negative for all ligands and ranged from ?1.74 to ?4.16 Kcal/mol. All enthalpies of binding were positive except butanal and hexanol. Change in free energy of binding per CH₂ in homologous aldehydes was greater for glycinin than β-conglycinin.     

Advances of Research on Glycinin and β-Conglycinin: A Review of Two Major Soybean Allergenic Proteins

Being an important crop, soybean is widely used in the world and plays a vital role in human and animal nutrition. However, it contains several antinutritional factors (ANFs) including soybean agglutinin, soybean protease inhibitors, soybean allergenic proteins, etc., that may result in poor food utilization, decreased growth performance, and even disease. Among these ANFs, soybean allergenic proteins can lead to allergic reactions in human and animals, which has become a public problem all over the world, but our knowledge on it is still inadequate. This paper aims to provide an update on the characteristics, detection or exploration methods, and in vivo research models of soybean allergenic proteins; especially glycinin and β-conglycinin are deeply discussed. Through this review, we may have a better understanding on the advances of research on these two soybean allergenic proteins. Besides, the ingredient processing used to reduce the allergenicity of soybean is also reviewed.     

Effects of Heat and Ionic Strength Upon Dissociation-Association of Soybean Protein Fractions

The effects of heat and ionic strength upon dissociation or association of soybean proteins were characterized by an immunoelectrophoresis. Among thermal products of both glycinin and β-conglycinin, the dissociated subunits of β-conglycinin retained their antigenic reactivities, while the thermal products of glycinin lost their antigenic reactivities. Appearance of immunoprecipitin arcs was dependent on the ionic strength of the solutions. This is based on the conformational changes at the ionic strengths below 0.1 where the thermal denaturation of β-conglycinin transforms from association to dissociation. The immunoelectrophoretic results can be used to determine the ionic strength of the solution in complex systems including whole soybean extracts and soybean milk. Relationship between the differences in textural properties of tofu and conformational changes of soybean protein is discussed.