植物钙调素及其结合蛋白的结构生物学进展

钙调素(CaM)作为植物中一种重要的钙受体蛋白,与Ca~(2+)结合调节细胞内一些靶蛋白(Ca MBPs)的活性,是细胞内Ca~(2+)信号传导途径中的主要信号转导分子,介导调控由Ca~(2+)引起的一系列生理生化反应。研究游离态CaM、结合了钙离子的Ca M以及靶蛋白肽–CaM复合体的结构有助于我们了解CaM介导Ca~(2+)信号传导途径的作用机理。综述了植物CaM的结构和性质,以及其与靶蛋白相互作用的研究进展。     

卵黄高磷蛋白磷酸肽在Caco-2细胞单层模型中的促钙转运作用

结合高温低压处理、胰蛋白酶和嗜热菌蛋白酶复合酶解制备具有高钙结合能力的卵黄高磷蛋白磷酸肽(Phosvitin phosphopeptides,PPP)。通过优化确定制备PPP-Ca的最佳条件为pH 9.5,多肽与钙质量比7∶1,此条件下钙螯合率达到97%,PPP-Ca在模拟胃肠道消化中显示了极高的稳定性。使用Caco-2细胞单层模型验证PPP-Ca在体外的钙转运作用,通过细胞形态、电阻值和荧光素钠透过率等验证Caco-2细胞模型,毒性实验(MTT)结果显示PPP-Ca对Caco-2细胞呈现低毒性作用。研究PPP-Ca和PPP对Caco-2细胞转运钙离子质量浓度和碱性磷酸酶(alkaline phosphatase,AKP)活性的影响,采用RT-PCR测定Calbindin D9K mRNA的相对表达量。结果表明,PPP和PPP-Ca能够显著提高AKP活性,钙转运量从(3.06±0.08)μg增到(5.66±0.62)μg,Calbindin D9K mRNA的表达量增加2倍,进而增强人体肠道中钙的转运吸收。研究结果为开发新的钙补充剂和卵黄高磷蛋白提供了科学证据。     

胶原三肽促钙吸收动物实验研究

采用"饲喂低钙饲料制备小鼠缺钙模型",以受试小鼠股骨指数、股骨钙含量和血清钙含量为检测指标,并与酪蛋白磷酸肽(CPP)进行比较,考察不同剂量胶原三肽(CTP)促钙吸收能力。结果表明,胶原三肽具备较好的促钙吸收功能,并且随剂量增加功能增强。     

食品成分对鱼鳞肽-钙复合物生物利用度的影响

鱼鳞肽具有一定的矿质离子结合能力,在钙离子存在的条件下,可与钙离子复合生成鱼鳞肽-钙复合物。研究采取Caco-2细胞模型与动物模型研究食品成分磷、VD3及植酸对鱼鳞肽-钙复合物生物利用度的影响。结果表明,食品中的磷可显著促进肽-钙复合物钙的吸收,提高其生物利用度,在Caco-2细胞与动物模型中,其钙吸收率分别提高了10.7%和39.8%(P0.05);VD3可促进无机钙的吸收,但对肽-钙复合物的吸收及转运无显著影响(P0.05);肽-钙复合物均可缓解食品成分植酸对钙离子的沉淀作用,提高其生物利用度。动物实验结果进一步证实,磷缺乏组大鼠钙表观吸收率及血清钙均低于正常组(P0.05),而VD3缺乏组和添加植酸对钙的吸收无显著影响。研究进一步证实,食品成分对无机钙与肽-钙复合物生物利用度的影响存在差异,推测与其不同的钙吸收及钙转运机制有关。     

Caco-2细胞模型用于乳源ACE抑制肽LL、LPEW的小肠吸收研究

通过建立并验证Caco-2细胞模型,分析血管紧张素转化酶(angiotensin-I converting enzyme,ACE)抑制肽LL、LPEW在小肠中的转运量,研究LL、LPEW的小肠吸收机制。从细胞形态、跨膜电阻和碱性磷酸酶活性3个方面验证Caco-2细胞模型可用性。分析ACE抑制肽LL、LPEW的Caco-2细胞转运量,LL的表观渗透系数(P_(app))为(275.17±8.28)×10~(-7 )cm/s,肠道吸收良好;LPEW的P_(app)为(5.13±1.49)×10~(-7) cm/s,相比于LL,肠道吸收量较低。加入旁路转运促进剂去氧胆酸钠、内吞抑制剂渥曼青霉素(Wortmannin)、肽转运载体竞争性抑制剂Gly-Pro,对比无抑制剂时LL的转运量,分析得到LL的跨膜转运机制可能为内吞途径。加入ATP能量生成抑制剂叠氮化钠、多药耐药蛋白抑制剂MK-571、P-糖蛋白抑制剂维拉帕米,对比无抑制剂时肽LL的转运量,得出LL没有外排作用,所以LL肠道吸收较好。     

联合补充维生素D、胶原肽和钙对成骨细胞发育的影响

目的:观察联合补充维生素D、胶原肽和钙对MC3T3-E1成骨细胞增殖及对骨保护素细胞内核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)、骨保护素(Osteoprotegerin,OPG)基因表达的影响。方法:α-MEM培养基培养MC3T3-E1细胞。检测Ca~(2+)(20μg/m L)、维生素D(0、10-12、10-11mol/L)、胶原肽(0、50、100μg/m L)三者交互作用剂量对MC3T3-E1细胞增殖的作用及RANKL、OPG mRNA表达。结果:维生素D、胶原肽和钙交互剂量作用下细胞增殖水平无明显差异。维生素D联合钙能够明显降低RANKL mRNA表达水平,提高OPG mRNA表达水平,降低RANKL/OPG比值。而胶原肽联合钙对RANKL以及OPG mRNA表达无明显影响。结论:维生素D联合钙可通过抑制小鼠成骨细胞RANKL mRNA表达、促进OPG mRNA表达,从而促进骨的形成,抑制骨的吸收。维生素D和钙联合补充胶原肽,对成骨细胞RANKL,OPG mRNA表达并无明显影响。     

白鲢鱼皮胶原蛋白肽钙螯合物的制备及其对Caco-2细胞钙转运的影响

利用食品工业加工副产物白鲢鱼皮,制备一种具有良好吸收性的新型补钙剂——胶原蛋白肽钙螯合物,并探究其在肠道细胞的钙转运吸收效果。以钙螯合率为指标,通过单因素正交试验确定肽钙螯合物的制备工艺,结果表明,螯合工艺确定为螯合时间60 min、温度60 ℃、pH8、肽钙质量比3:1,最终螯合率可达68.10%。傅里叶红外光谱显示Ca~(2+)与胶原蛋白肽的氨基和羰基相结合生成肽钙螯合物;X射线衍射显示,肽与钙螯合后,从不规则的非晶体结构转变为规则的晶体结构。体外模拟消化试验证明,肽钙螯合物具有良好的抗消化性,经过体外模拟消化后,仍保留了64.8%的持钙能力。在Caco-2细胞单层模型中,与CaCl_2组相比,肽钙螯合物具有显著钙吸收活性(P0.05)。研究结果为白鲢鱼皮的高值利用和新型钙补充剂的开发提供了科学依据。     

三文鱼骨胶原低聚肽钙增加SD大鼠的骨密度

以三文鱼骨为原料制备三文鱼骨胶原低聚肽,与钙离子螯合为三文鱼骨胶原低聚肽钙并作为受试物,以低中高剂量作用SD大鼠,并设置空白组和碳酸钙阳性对照组,检测体重、钙吸收率与储留率、股骨干重、股骨长、骨密度等重要指标,评价三文鱼骨胶原低聚肽钙的增加骨密度功能。结果显示,螯合物的总蛋白含量为65.37%,螯合率为52.56%,螯合物得率为43.12%;各试验组体重无显著性差异(p>0.05),中、高剂量组和碳酸钙组的钙吸收率分别为39.18%、40.52%、38.38%,储留率分别为38.80%、39.27%、37.58%,显著高于空白对照组36.31%,35.91%(p<0.05),且高剂量组明显优于同等钙含量的碳酸钙组(p<0.05);另外高剂量组的股骨干重、股骨中点直径分别为0.57(g)和2.40(mm),股骨近端、远端和中点密度分别为0.19、0.22、0.17(g/cm2),均高于空白组(p<0.05),与碳酸钙组无显著性差异。表明三文鱼骨胶原低聚肽钙具有良好的促钙吸收效果和增加骨密度的功能,为相关新型功能性食品的开发利用奠定良好的理论基础。     

花生蛋白钙螯合肽的制备、富集及表征

本文对花生蛋白钙螯合肽的制备工艺进行了研究,并对所得水解产物及其钙螯合物进行了表征。结果表明,在6种市售的蛋白酶中,Alcalase 2.4L对花生蛋白的水解能力最强,其与ProteAXH进行分步复合水解可进一步提高水解效果,在最适条件下所得花生肽的钙结合能力达到了84.09 mg/g、蛋白质回收率高达87.89%;采用酸法脱酰胺可显著提高花生肽的钙结合能力,在最适条件下可达到132.04 mg/g;利用大孔树脂DM301对脱酰胺花生肽进行富集可进一步提高其钙结合能力,在最优条件下可高达164.34 mg/g。荧光光谱及圆二色谱分析证实花生肽与钙离子发生了螯合作用,红外光谱分析则表明花生肽主要通过其羧基与钙离子进行结合。因此,花生肽是一种潜在的钙螯合剂,这对于花生蛋白的高值化综合利用具有重要的参考价值。     

食源性肽的功能特性评价方法、分子构效关系及吸收代谢机制

肽是分子结构介于氨基酸和蛋白质之间的化合物,具有易吸收、低渗透压、低免疫原性、高组织渗透性等营养特性,可调控细胞代谢,是组织细胞的重要化学信使。食源性肽是食物蛋白质经限制性酶解法或生物发酵法等制备的重要营养基料,兼具良好的营养价值和多种生理调节活性。但目前食源性肽转运吸收过程、胞内亚细胞器时空动态分布以及营养代谢调控功能尚待研究,且不同结构和功能的肽分子(跨膜肽、信号肽、转运肽等)间协同作用模式也未明晰,严重制约了食源性肽的基础研究与产业快速发展。系统介绍了以化学、体外生物、低等/高等模式动物以及临床等多维度的肽功能评价方法;解析了食源性肽的分子构效关系,以“自上而下”与“自下而上”的2种方式分别阐述了食源性肽的分子构效规律分析方法,并创新了基于分子杂化轨道的“结构-功效”表征理论;揭示了不同分子结构特征(一级氨基酸序列、空间构型、主链肽键的排列方式、侧链官能团的多样化及α-碳的手性等)的肽分子在转运吸收过程中的构效关系;进一步探讨了营养研究领域中复杂体系下食源性蛋白肽吸收及调控机体代谢的规律,深度挖掘肽吸收代谢与营养的科学基础,对基于食源性肽开展精准营养调控具有重要借鉴意义。对食源性肽的功能特性评价方法、“构-效”关系和吸收代谢机理进行了系统的阐述,以期为未来食源性肽的研究提供科学的指导和参考。     

Gene mapping,gene-set analysis,and genomic prediction of postpartum blood calcium in Holstein cows

The onset of lactation results in a sudden irreversible loss of Ca for colostrum and milk synthesis. Some cows are unable to quickly adapt to this demand and succumb to clinical hypocalcemia, whereas a larger proportion of cows develop subclinical hypocalcemia that predisposes them to other peripartum diseases. The objective of this study was to perform a comprehensive genomic analysis of blood total Ca concentration in periparturient Holstein cows. We first performed a genomic scan and a subsequent gene-set analysis to identify candidate genes, biological pathways, and molecular mechanisms affecting postpartum Ca concentration. Then, we assessed the prediction of postpartum Ca concentration using genomic information. Data consisted of 7,691 records of plasma or serum concentrations of Ca measured in the first, second, and third day after parturition of 959 primiparous and 1,615 multiparous cows that calved between December 2015 and June 2020 in 2 dairy herds. All cows were genotyped with 80k SNPs. The statistical model included lactation (1 to 5+), calf category (male, females, twins), and day as fixed effects, and season-treatment-experiment, animal, and permanent environmental as random effects. Model predictive ability was evaluated using 10-fold cross-validation. Heritability and repeatability estimates were 0.083 (standard error = 0.017) and 0.444 (standard error = 0.028). The association mapping identified 2 major regions located on Bos taurus autosome (BTA)6 and BTA16 that explained 1.2% and 0.7% of additive genetic variance of Ca concentration, respectively. Interestingly, the region on BTA6 harbors the GC gene, which encodes the vitamin D binding protein, and the region on BTA16 harbors LRRC38, which is actively involved in K transport. Other sizable peaks were identified on BTA5, BTA2, BTA7, BTA14, and BTA9. These regions harbor genes associated with Ca channels (CACNA1S, CRACR2A), K channels (KCNK9), bone remodeling (LRP6), and milk production (SOCS2). The gene-set analysis revealed terms related to vitamin transport, calcium ion transport, calcium ion binding, and calcium signaling. Genomic predictions of phenotypic and genomic estimated breeding values of Ca concentration yielded predictive correlations up to 0.50 and 0.15, respectively. Overall, the present study contributes to a better understanding of the genetic basis of postpartum blood Ca concentration in Holstein cows. In addition, the findings may contribute to the development of novel selection and management strategies for reducing periparturient hypocalcemia in dairy cattle.     

Effects of peptide–calcium complexes from sunflower seeds and peanuts on enhancing bone mineral density

Hydrolysed proteins from sunflower seed and peanut were separated by ultrafiltration. Results showed that 1–3 kDa sunflower seed peptide (SSP₄) and ≥10 kDa peanut peptide (PP₁) fractions had the highest Ca-binding capacity of 104.80 and 124.74 mg g⁻¹, respectively. After Ca binding, scanning electron microscopy results showed that the loose sheet-like structure of SSP₄ and PP₁ transformed into a granular structure. Fourier-transform infrared spectroscopy results showed that carboxyl and amide groups were involved in Ca binding. Growing male mice were assigned to eight groups: control, CaCO₃ and six experimental groups (low, medium and high doses for each SSP₄-Ca and PP₁-Ca). Apparent Ca absorption rate, bone mineral density (BMD), bone surface microstructure and biomechanical index and serum biochemical index were evaluated. Animal experiment results indicated that SSP₄-Ca and PP₁-Ca can improve Ca bioavailability; effectively enhance BMD and structure; and promote Ca absorption. The peptide–Ca complexes were absorbed better than CaCO₃ and promoted Ca accumulation in the bone. PP₁-Ca was better than SSP₄-Ca in promoting calcium absorption and enhancing BMD.     

多肽螯合钙的研究进展

钙作为一种必需营养素,在人体中有着极为重要的作用。现如今,补钙制剂在市场上占有极大的份额,但以无机钙和有机钙为主的第一、第二代补钙制剂仍有较大缺陷,吸收率和生物利用率低,而多肽螯合钙作为第三代具有活性结构的生物钙补钙制剂稳定性好、抗干扰能力强、吸收效果好、生物利用度高,因此越来越受到关注。该文对多肽螯合钙的结合机制、制备方法、吸收途径、生物利用度等方面的研究进行综述,以期对补钙制剂的研究及开发提供新思路。     

Cloning, characterization and expression analysis of calcium channel beta subunit from pearl oyster (Pinctada fucata)

The absorption, transport and localization of calcium underlie the basis of biomineralization, and Ca(2+) entry into epithelial cell is the primary step in shell formation. However, the related mechanism of Ca(2+) transport is poorly documented at the gene or protein level. L-type voltage-dependent calcium channels may be involved in calcium transport for biomineralization in some marine invertebrates. In this study, a full-length cDNA of a voltage-dependent calcium channel beta subunit from Pinctada fucata (PCabeta) was cloned, and its amino acid sequence was deduced. PCabeta shared 51%-67% apparently sequence identity with voltage-dependent calcium channel beta subunits from other species. However, PCabeta was much shorter than other voltage-dependent calcium channel beta subunits particularly at the carboxyl terminus, indicating that it is likely a truncated beta subunit isoform. Semi-quantitative RT-PCR analysis showed that PCabeta was expressed in all the tested tissues and that it had a higher expression level in the gill tissue and hemolymph than in other tissues, suggesting that L-type voltage-dependent calcium channels are responsible for Ca(2+) absorption in the gill and Ca(2+) entry into hemocytes. In the mantle, PCabeta mRNA was predominantly expressed in the inner and middle folds of the mantle epithelium, suggesting that L-type voltage-dependent calcium channels are involved in Ca(2+) absorption from the ambient medium in the mantle. All these results suggest that voltage-dependent calcium channels are involved in Ca(2+) uptake and transport during oyster biomineralization.     

大黄鱼蛋白肽-钙螯合物的制备及性质研究

目的 制备大黄鱼蛋白肽-钙螯合物,并研究其结构性质。方法 以大黄鱼鱼糜为原料,通过胃蛋白酶、胰蛋白酶、中性蛋白酶进行酶解制备多肽,将肽与钙进行螯合,探究大黄鱼蛋白肽及其肽-钙螯合物的性质功能。结果 3种酶解肽中,胰酶肽钙螯合含量最高,胰酶肽的粒径最小,肽-钙螯合物相比于肽的粒径都有所减小,说明肽-钙螯合物是一种致密的纳米颗粒并呈现出折叠多孔的网络结构。这些变化可归因于肽和钙离子之间的相互作用。紫外吸收光谱、荧光光谱、红外图谱和圆二色光谱的结果表明大黄鱼蛋白肽与钙离子螯合,生成了新的肽-钙螯合物。结论 3种酶解肽中,胰蛋白酶水解物的钙离子结合能力在3种蛋白酶获得的水解物中最高。因此,在本研究中胰蛋白酶被认为是制备大黄鱼蛋白水解物钙离子结合活性肽的最优酶制剂,为大黄鱼的高值化利用和新型钙制剂的开发提供了理论依据和实验参考。     

补钙面条加工技术研究

对添加胶原多肽螯合钙的补钙面条加工技术进行了研究。以猪皮明胶为原料,制备胶原多肽螯合钙,并对其结构进行了表征。添加胶原多肽螯合钙、猪皮明胶、复合磷酸盐和食盐对面条品质进行改良,通过感官评价对补钙面条的加工条件进行优化。试验结果表明,补钙面条的最佳配方为:胶原多肽螯合钙添加量为2%,猪皮明胶添加量为1.5%,复合磷酸盐添加量为0.8%,食盐添加量为1.5%,小麦粉94%。面条产品弯曲断条率为0,熟断条率为0,干物质失落率为6.9%,蒸煮吸水率为167.5%,面汤浊度为0.104。经感官评价,面条得分为88.7分。通过添加胶原多肽螯合钙,补钙面条中强化钙量为143.6 mg/100 g。补钙面条外观色泽良好,风味较好,食用营养价值高。     

蛋清肽- 钙配合物体内促钙吸收作用研究

目的：为了考察蛋清肽-钙配合物(EWP-Ca)在动物体内的补钙效果。方法：将60只Wistar雄性大鼠随机分为6组：普通组、缺钙模型组、CaCO3组以及低、中、高剂量EWP-Ca组,通过构造缺钙大鼠模型,进行了为期30d的补钙实验,实验结束后对各种与补钙相关的指标进行测定。结果：与缺钙模型组相比,高剂量EWP-Ca组(Ca剂量为106.4mg/(kg ·d))能极显著增加大鼠股骨直径、干质量、骨钙含量及股骨密度(P＜0.01),极显著降低血清碱性磷酸酶(AKP)活力(P＜0.01);与钙含量相同的CaCO3组相比,EWP-Ca高剂量组股骨直径、骨钙含量极显著和显著增加(P＜0.01,P＜0.05)。结论：EWP-Ca具有体内促进钙吸收作用,且效果优于钙含量相同的CaCO3。     

酪蛋白钙肽研究进展

酪蛋白钙肽是含磷酸丝氨酸残基具生理功能多肽,该文介绍酪蛋白钙肽物理特性,生理功能,制备工艺,检测方法及在乳制品中应用。     

A Newly Isolated Ca Binding Peptide from Whey Protein

In this study, whey protein was hydrolyzed by trypsin. A calcium binding peptide was isolated and purified by ultra-filtration, gel filtration chromatography, and reverse phase HPLC from the hydrolysate. The target peptide, with a molecular weight of 555.54 Da and with the amino acid sequence Ser-Thr-Glu-Tyr-Gly, corresponds to the 48 to 52 th amino acid sequence of bovine α-lactalbumin, which exhibited a significant effect on promoting the absorption of calcium in mice. The peptide could be used as a food additive to prevent calcium deficiency in humans.