Calcium-chelating peptides from rabbit bone collagen:characterization,identification and mechanism elucidation

This study aimed to characterize and identify calcium-chelating peptides from rabbit bone collagen and explore the underlying chelating mechanism.Collagen peptides and calcium were extracted from rabbit bone by instant ejection steam explosion(ICSE)combined with enzymatic hydrolysis,followed by chelation reaction to prepare rabbit bone peptide-calcium chelate(RBCP-Ca).The chelating sites were further analyzed by liquid chromatography-tandem mass(LC-MS/MS)spectrometry while the chelating mechanism and binding modes were investigated.The structural characterization revealed that RBCP successfully chelated with calcium ions.Furthermore,LC-MS/MS analysis indicated that the binding sites included both acidic amino acids(Asp and Glu)and basic amino acids(Lys and Arg),Interestingly,three binding modes,namely Inter-Linking,Loop-Linking and Mono-Linking were for the first time found,while Inter-Linking mode accounted for the highest proportion(75.1%),suggesting that chelation of calcium ions frequently occurred between two peptides.Overall,this study provides a theoretical basis for the elucidation of chelation mechanism of calcium-chelating peptides.

Progress in the extraction and application of collagen peptides

Collagen peptide is the product of complete hydrolysis of collagen,which has a relatively small molecular weight and is more easily absorbed than proteins and amino acids.Collagen peptide not only has unique nutritional value,but also has certain physiological functions,which makes it has great potential value in various fields,so it has set off a wave of research on collagen peptide in the biological world.This paper describes the sources and extraction methods of collagen peptides,and describes the research progress and application of collagen peptides in the medical,food,material and skin care industries according to their physiological functions,which will provide new ideas for the future research of collagen peptides.

Anti-skin aging effects and bioavailability of collagen tripeptide and elastin peptide formulations in young and middle-aged women

Background:Collagen peptides(CP),including tripeptides and elastin peptides(EP),are known for their in vitro and in vivo anti-skin aging effects.Despite positive results in animal models,the combination effects of CP and EP and the bioavailability of CP in human studies,particularly in young and middle-aged women,remain underexplored.Objective:To evaluate the effects of an orally administered collagen drink combining CP and EP on the skin health of young and middle-aged women.Materials and Methods:A single-center,randomized,double-blind,parallel-controlled trial was conducted,utilizing the WONDERLABR fish collagen tripeptide beverage.Participants consumed the drink over an 8-week period.Results:Compared to the placebo group,the collagen drink group showed significant improvements in skin hydration(39.19%increase),transepidermal water loss(33.45%decrease),skin elasticity(25.37%increase),dermal collagen content(21.64%increase),pore size(7.94%decrease),wrinkle length(18.09%decrease),skin smoothness(2.85%improvement),and skin roughness(15.32%decrease).Overall pore volume decreased by 60%,and visual assessments indicated a decrease in skin luminosity by 15.20%and smoothness index by 22.55%.Mass spectrometry demonstrated a significant increase in collagen efficacy components,including blood pH and GPH levels(P<0.05).Conclusion:The study confirmed the combination nourishing and anti-skin aging effects of EP and CP on the skin of young and middle-aged women,demonstrating significant improvements in various skin parameters and good bioavailability of collagen peptides.

Review on marine collagen peptides induce cancer cell apoptosis,necrosis,and autophagy by reducing oxidized free radicals

Marine collagen peptides(MCPs)are natural products prepared by hydrolyzing marine collagen protein through a variety of chemical methods or enzymes.MCPs have a range of structures and biological activities and are widely present in marine species.MCPs also have a small molecular weight,are easily modified,and absorbed by the body.These properties have attracted great interest from researchers studying antioxidant,anti-tumor,and anti-aging activities.MCPs of specific molecular weights have significant anti-tumor activity and no toxic side effects.Thus,MCPs have the potential use as anti-cancer adjuvant drugs.Free radicals produced by oxidation are closely related to human aging,cancer,arteriosclerosis,and other diseases,but their relationship with cancer is not well known.In this review,we focus on the antioxidant properties of MCPs in the treatment of cancer,highlighting their antioxidant molecular structure and potential for clinical practice.

Active Peptides and Motifs within Collagen and Other ECM Proteins

Collagens are the most abundant proteins in mammals and form an extracellular matrix (ECM) with other components as the structural support of muscle, skin, corneas and blood vessels etc. Other than providing structural support, the ECM exhibits active communication with cells and influences many cellular processes including migration, wound healing, differentiation and cancer metastasis. Though collagen proteins contain highly repetitive primary sequences and defined tertiary structures, more and more studies have shown that many short peptides/motifs within collagen proteins play key roles in various biological processes. These short sequences are effective within triple helical structures or independently as stand-alone molecules resulting from proteolytic degradation. Besides endogenous ECM-derived peptides, many more functional peptides have been produced by tissue processing, chemical synthesis, and recombinant protein production. In this review, we summarize different peptides/motifs identified in collagen and other ECM proteins and discuss their potential for medical, personal care, and cosmetics applications.

Effect of Marine Collagen Peptides on Markers of Metabolic Nuclear Receptors in Type 2 Diabetic Patients with/without Hypertension

Objective To explore Effects of marine collagen peptides （MCPs） on markers of metablic nuclear receptors, i.e peroxisome proliferator-activated receptor （PPARs）, liver X receptor （LXRs） and farnesoid X receptor （FXRs） in type 2 diabetic patients with/without hypertension. Method Study population consisted of 200 type 2 diabetic patients with/without hypertension and 50 healthy subjects, all of whom were randomly assigned to MCPs-treated diabetics （n=50）, placebo-treated diabetics （n=50）, MCPs-treated diabetics with hypertension （n=50）, placebo-treated diabetics with hypertension （n=50）, and healthy controls （n=50）. MCPs or placebo （water-soluble starch） were given daily before breakfast and bedtime over three months. Levels of free fatty acid, cytochrome P450, leptin, resistin, adiponectin, bradykinin, NO, and Prostacyclin were determined before intervention, and 1.5 months, and 3 months after intervention. Hypoglycemia and the endpoint events during the study were recorded and compared among the study groups. Result At the end of the study period, MCPs-treated patients showed marked improvement compared with patients receiving placebo. The protection exerted by MCPs seemed more profound in diabetics than in diabetics with hypertension. In particular, after MCPs intervention, levels of free fatty acid, hs-CRP, resistin, Prostacyclin decreased significantly in diabetics and tended to decrease in diabetic and hypertensive patients whereas levels of cytochrome P450, leptin, NO tended to decrease in diabetics with/without hypertension. Meanwhile, levels of adiponectin and bradykinin rose markedly in diabetics following MCPs administration. Conclusion MCPs could offer protection against diabetes and hypertension by affecting levels of molecules involved in diabetic and hypertensive pathogenesis. Regulation on metabolic nuclear receptors by MCPs may be the possible underlying mechanism for its observed effects in the study. Further study into its action may shed light on development of new drugs based on bioactive peptides from marine sources.

Comparative Study on the Antioxidant Activity of Peptides from Pearl Oyster(Pinctada martensii) Mantle Type V Collagen and Tilapia(Oreochromis niloticus) Scale Type Ⅰ Collagen

In this study, Pearl oyster mantle type V collagen(POMC) and tilapia scale type I collagen(TSC) were extracted and hydrolyzed by various proteases in order to obtain peptides. The antioxidant activity of the peptides was investigated by DPPH, hydroxyl radical scavenging experiments and a dynamic digestion model in vitro. The results show that there are significant differences in amino acid composition between POMC and TSC. The collagen peptides obtained from pearl oyster mantle(POMCP) by treating with alkaline protease exhibited higher antioxidant activity than that from tilapia scale(TSCP) treated with papaya protease, and both of them showed greater DPPH and hydroxyl radical scavenging activity than other peptides. After being separated via Sephadex G-25 chromatography, the M1 fraction isolated from POMCP, and the S1 fraction from TSCP with which both had higher molecular weights showed the strongest antioxidant activity than other fractions, and the M1 fraction exhibited stronger antioxidant activity than the S1 fraction in scavenging free-radicals and protecting cells from the oxidation damage. Furthermore, after treating the dynamic digestion system model in vitro, the DPPH and hydroxyl radical scavenging activity of the M1 fraction increased slightly. These results suggest that POMCP exhibits stronger antioxidant activity than TSCP, which means that PMOP may be a good candidate to be a potential natural antioxidant in the food-processing industry.

Recent progress in preventive effect of collagen peptides on photoaging skin and action mechanism

Ultraviolet(UV)-induced photoaging skin has become an urgent issue.The functional foods and cosmetics aiming to improve skin photoaging are developing rapidly,and the demand is gradually increasing year by year.Collagen peptides have been proven to display diverse physiological activities,such as excellent moisture retention activity,hygroscopicity,tyrosinase inhibitory activity and antioxidant activity,which indicates that they have great potential in amelioration of UV-induced photoaging.The main objective of this article is to recap the main mechanisms to improve photoaging skin by collagen peptides and their physiological activities in photo-protection.Furthermore,the extraction and structural characteristics of collagen peptides are overviewed.More importantly,some clinical trials on the beneficial effect on skin of collagen peptides are also discussed.In addition,prospects and challenges of collagen peptides are emphatically elucidated in this review.This article implies that collagen peptides have great potential as an effective ingredient in food and cosmetics industry with a wide application prospect.

Study on the Reparative Effect of Peptide RW3 Binding Active Collagen after Photoelectric Skincare

The photoelectric microneedle treatment instrument is also widely used due to the rapid development of medical cosmetology in China in recent years. It also causes a lot of skin discomfort after the consumers carry out such projects. This study, which combines small molecule active peptides (RW3) and active collagens and imitates the photoelectric treatment through in vitro and in vivo experiment, finds that small molecule active peptides (RW3) and active collagens have different improvement effects on cell proliferation, migration, anti-UV damage, inhibition of ear swelling and inflammation in mice, repair of UV damage and skin damage caused by microneedles. .

The Effectiveness of Specific Collagen Peptides on Osteoarthritis in Dogs-Impact on Metabolic Processes in Canine Chondrocytes

In clinical trials over the past decade, the beneficial effect of orally administered collagen peptides in osteoarthritic dogs has been clearly demonstrated [1] [2] [3]. Although a statistically significant improvement in the lameness and vitality of dogs in general has been documented, the mode of action of the collagen peptide treatment is still under discussion. A previous study [3] indicated that the reduction in lameness and increased mobility in dogs after collagen peptide treatment were associated with a statistically significantly lowered plasma content of MMP-3, which is involved in collagen degradation. In addition, the content of the MMP-antagonist TIMP-1 increased slightly after collagen peptide supplementation, suggesting a direct impact on the cartilage metabolism, particularly on the decrease of extracellular matrix degradation. Based on these findings, the impact of specific collagen peptides (PETA-GILE?) on cartilage metabolism was tested in canine chondrocytes in the current investigation. In addition to the biosynthesis of various matrix molecules (type II collagen, aggrecan and elastin), the RNA profile of inflammatory cytokines and degenerative matrix molecules was investigated. The results showed clearly that the supplementation of specific collagen peptides reduced catabolic processes, as indicated by a statistically significant decrease in inflammatory cytokines and proteases in canine chondrocytes compared with untreated control experiments. In addition, a statistically significantly enhanced biosynthesis of type II collagen, elastin, and aggrecan was observed. Hence, the current data supports the suggested anti-inflammatory effect of specific collagen peptides, but also clearly demonstrates a pronounced stimulatory impact on matrix molecule synthesis. A combination of both observed effects might help to explain the previously reported clinical improvements after collagen peptide supplementation. Furthermore, the beneficial effect of the specific collagen peptides was also confirmed in case reports on osteoarthritic dogs that demonstrated decreased lameness and increased vitality in the affected animals after PETAGILE treatment.

Effects of Collagen Peptide from Cyanea nozakii on Mouse Immune Function

[Objective ] The study aimed to provide a theoretical basis for the clinical application of collagen peptide from Cyanea nozakii. [ Method] After acute toxicity test on mice, collagen peptide from C. nozakii were given to mice by continuous intragastric administration for 30 d at the doses of 25, 50, 100 mg/kg, and then the phagocytosis of macrophage, delayed type hypersensitivity （DTH） and serum hemolysin level were determined. [ Result] Collagen peptide from C. nozakii was atoxic or low toxic, and the three immune indices of experimental groups were signifi- canUy higher than those of the control group （treated with same volume of normal saline） at 0.05 or 0.01 level. E Conclusion] Collagen peptide from C. nozakii has a certain immunopotentiation.

Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates

A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.

Supplementation with yak (Bos grunniens) bone collagen hydrolysate altered the structure of gut microbiota and elevated short-chain fatty acid production in mice

In this study, yak bone collagen hydrolysate(YBCH)was produced by mixed proteases and provided to standard-diet mice at a different dose(low dose(LD), medium dose(MD), and high dose(HD))to investigate its effects on the composition of gut microbiota and short-chain fatty acids(SCFA)production. It was found that YBCH was mainly composed of small molecular peptides whose molecular weight below 2 000 Da. Notably, supplementation with different doses of YBCH could significantly downregulate the ratio of Firmicutes to Bacteroidetes in the fecal microbiota. At the family level, the Lachnospiraceae abundance was significantly reduced in the YBCH gavage groups(mean reduction ratio 41.7 %, 35.2%, and 36.4% for LD, MD, and HD group, respectively). The predicted functions of gut microbes in the MD group were significantly increased at “lipid metabolism” and “glycan biosynthesis and metabolism”. Moreover, the SCFA production in the YBCH groups was elevated. Especially, the concentration of acetic acid, propionic acid, and butyric acid in the MD group was separately increased 79.7%, 89.2%, and 78.8% than that in the NC group. These results indicated that YBCH might be applied in the development of functional food for intestinal microecological regulation.

Structural, thermal and enzymatic analysis of naturally occurring and D-amino acid substituted peptides

Designing of new peptide materials for biomedical and protein engineering applications are important. In the present work an attempt has been made to study the effect of D-Leu in collagen like tetra peptide on the structure and stability of peptide against enzymes and results are compared with its chiral counterpart L-form. Effect of replacement of L-Leu in Leu-Gly-Pro-Ala tetra peptide with D-Leu on structure has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, D-Leu substitution leads to conformational changes in Leu-Gly-Pro-Ala secondary structure from β-sheet to turns. L → D-Leu Configurational changes in Leu-Gly-Pro-Ala owes to enhanced thermal stability which has been substantiated through CD and differential scanning calorimetry. Change in chirality of the leucine inhibits collagenolytic activity, which enables to design selective inhibition of proteases with greater specificity.

A novel anti-inflammatory and immunomodulatory drug CP-25 alleviated collagen induced arthritis by down-regulating BAFF-NF-κκB signaling pathway

OBJECTIVE To investigated the regulatory effect of paeoniflorin-6′-O-benzene sulfonate(CP-25) on B cell activating factor(BAFF)/BAFF receptor-nuclear factor of kappa B(NF-κB) signaling in B cell of collagen induced-arthritis(CIA) mice.METHODS Mice CIA was induced by injection of typeⅡcollagen(CⅡ).The arthritis index(AI) and swollen joint count(SJC) were assessed,and histopathology of spleen and joints were observed.The percentage of B cells subsets,BAFF receptor expressions were analyzed by flow cytometry.BAFF and immunoglobulin(Ig) levels were measured by protein antibody array.The expressions of TRAF2,MKK3,MKK6,p-P38,and p-NF-κB65 in NF-κB signaling mediated by BAFF were analyzed by western blot.RESULTS CP-25 decreased AI and SJC,restored abnormal weights,reduced thymus index and spleen index,inhibited T/B cells proliferation,alleviated the histopathology of spleen and joints in CIA mice.CP-25 also reduced high levels of serum BAFF and immunoglobulin,decreased CD19+B cells,CD19+CD27+B cells,and CD19-CD27+CD138+plasma cells,inhibited BAFFR and TACI expressions,decreased the expressions of TRAF2,MKK3,MKK6,p-P38,and p-NF-κB65.Compared with biological agents etanercept and rituximab,CP-25 restored high T cells proliferation and percentages of B subsets to normal level,and recovered the high levels of IgA,IgD,IgG1,IgG2 a and high expressions molecules in NF-κB signaling to normal levels.The action intensity of rituximab and etanercept was more strong than CP-25.The inhibitor effects of rituximab and etanercept on AI and SJC,thymus index,proliferation of T cells and B cells subsets were strong,and down-regulated the indexes to under normal levels.CONCLUSION CP-25 might be a promising anti-inflammatory immune and regulation drug,which alleviated CIA and regulated the functions of B cells through BAFF/BAFF receptor-NF-κB signaling.

碱性蛋白酶酶解蛋白制备活性肽的质控研究

以胶原蛋白和大豆分离蛋白为主要研究对象,应用体积排阻色谱(SEC)结合多角度激光光散射(MALLS)及示差折光(RI)联用技术对碱性蛋白酶酶解蛋白制备肽段的过程进行了详细分析。在酶解过程中,不同来源的胶原蛋白(牛、鸡)和大豆分离蛋白在分子量分布、体系的分散度及粒径变化规律上表现出了典型的差异。结果表明,牛胶原蛋白在酶解开始时就迅速被降解产生了大量小分子肽段,鸡胶原蛋白相对于牛胶原蛋白在相同的酶解条件下更难产生小分子肽段,大豆分离蛋白随着酶的加入则是表现出了先聚集再降解的特性。SEC-MALLS技术可以有效把控蛋白酶解的过程以及体系中肽段分子量分布,该技术可以为活性肽产品的开发及品质检测提供依据。

超声辅助酶解制备草鱼鳞胶原肽的特征风味解析

该研究以超声辅助单酶酶解组/分步酶解组的胶原肽为研究对象,传统的单酶酶解组和分步酶解组为对照,通过电子鼻、顶空固相微萃取结合气相色谱-质谱联用技术、超高效液相色谱仪、氨基酸分析仪和电子舌对4组胶原肽的气味特征和滋味特征进行了分析鉴定;同时,对4组样品分别进行了感官评价。结果表明,超声组挥发性风味物质的含量和种类均有所提升,其中超声辅助单酶酶解组种类由20种提高至24种,含量由5 966.70μg/kg提高至6 422.62μg/kg,超声辅助分步酶解组种类由22种提高至24种,含量由6 202.30μg/kg提高至7 480.43μg/kg,同时,超声组呈味物质的含量也有所上升,呈鲜味的肌苷酸和鸟嘌呤核苷酸含量均有所提高,苦味氨基酸和鲜味氨基酸含量也有所提高,即超声会影响挥发性风味物质和滋味物质核苷酸和游离氨基酸的种类和含量,在一定程度上可以改善胶原肽的气味特征和滋味品质。此外,感官评价结果表明,超声组整体风味轮廓较好,鲜味有所提升,腥味/异味有所降低,为超声辅助酶解工艺制备胶原肽的风味研究提供了支持。

基于Lasso回归模型研究抗冻肽性质对冷冻鱼糜肌原纤维蛋白的影响

为探究抗冻肽性质对其保护冷冻鱼糜肌原纤维蛋白效果的影响,本实验对3种抗冻肽(丝胶肽、胶原肽及鲢鱼酶解产物)的性质进行表征,分析不同冻藏条件下鱼糜肌原纤维蛋白指标变化规律,并基于最小绝对缩小和选择算子(least absolute shrinkage and selection operator,Lasso)回归构建了“冻藏工艺-抗冻肽性质-肌原纤维蛋白指标”的关联模型。结果表明,所用的3种抗冻肽均为分子质量低(m<10000 Da)、亲水性强(静态接触角小于30°)的“超活性”抗冻肽(热滞活性大于0.60℃),极性氨基酸占比超过60%;所用抗冻肽能够显著减缓冻藏处理对肌原纤维蛋白的负面影响;冻藏处理后,抗冻肽组鱼糜肌原纤维蛋白含量平均值维持在50.44~54.47 mg/g,Ca^(2+)-ATPase活性平均值为1.61~1.67 U/mg,表面疏水性指数平均值相比新鲜鱼糜仅增长39.91%~42.40%,肌原纤维蛋白变性速率远低于未添加抗冻剂组。Lasso回归模型解释了抗冻肽性质对冷冻鱼糜肌原纤维蛋白变化的影响,证明低分子质量、高热滞活性的抗冻肽有助于减缓肌原纤维蛋白含量、Ca^(2+)-ATPase活性、表面疏水性及特征指数的劣变速率;抗冻肽亲/疏水性的区别导致其与冰晶和蛋白之间的相互作用不同,从而对鱼糜肌原纤维蛋白产生不同影响。本研究可为抗冻肽在冷冻鱼糜中的应用及鱼糜制品的品质调控提供理论基础,同时为研究新型高效的抗冻剂提供方向。

胶原蛋白肽/硅橡胶复合材料亲水性研究

利用双辊开炼机将胶原蛋白肽加入硅橡胶中,制备胶原蛋白肽/硅橡胶复合材料。结果表明:胶原蛋白肽在复合材料中有结构控制剂和延迟硫化的作用;随着胶原蛋白肽用量的增加,复合材料的硬度增加,100%定伸应力从1.92 MPa增至2.66 MPa;拉伸强度和撕裂强度降低。当复合材料中胶原蛋白肽含量为4份时,复合材料的最大热失重速率温度比纯硅橡胶降低1.6%,说明加入胶原蛋白肽,降低了复合材料热稳定性;复合材料红外光谱中出现了硅橡胶和胶原蛋白肽的特征峰;接触角比纯硅橡胶降低了4.5%,说明复合材料的亲水性有一定改善。

沙丁鱼鱼鳞胶原蛋白肽的制备及其抗氧化活性研究

目的优化沙丁鱼(Sardinapilchardus)鱼鳞胶原蛋白肽的制备工艺和评价不同分子量大小胶原蛋白肽的抗氧化活性。方法以沙丁鱼鱼鳞为原料,通过酶工程结合单因素与响应面实验优化胶原蛋白肽制备工艺,采用超滤膜分级分离鱼鳞胶原蛋白肽,并通过体外自由基化学体系分析对比不同分子量大小(10、5、1 kDa)的胶原蛋白肽的抗氧化活性。结果采用碱性蛋白酶进行酶解鱼鳞,胶原蛋白肽的得率最高;最优酶解工艺参数为:碱性蛋白酶加酶量7526 U/g、料液比1:20(g/mL)、pH 10.5、酶解温度67℃、酶解时间4 h,此条件下鱼鳞胶原蛋白肽得率最高,达43.44%±1.59%。通过超滤获得的分子量小于1k Da的鱼鳞胶原蛋白肽对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和羟基自由基(hydroxyl radical,·OH)的清除能力最强,其半抑制浓度(half maximal inhibitory concentration,IC_(50))分别为0.72 mg/mL和1.76 mg/mL;分子量为1~5 kDa的鱼鳞胶原蛋白肽对超氧阴离子自由基(superoxide radical,O^(2-)·)的清除效果最好,其IC50为1.45 mg/mL。结论碱性蛋白酶能够有效制备鱼鳞胶原蛋白肽,且小于1 kDa的鱼鳞胶原蛋白肽抗氧化活性最好。