Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins

Novel bioactive peptides from camel milk protein hydrolysates (CMPH) were identified and tested for inhibition of cholesterol esterase (CEase), and their possible binding mechanisms were elucidated by molecular docking. Papain-generated CMPH showed the highest degree of hydrolysis. All CMPH produced upon enzymatic degradation demonstrated a dramatic enhancement of CEase inhibition compared with intact camel milk proteins, with papain-generated hydrolysate P₉ displaying the highest inhibition. Peptide identification and their modeling through PepSite 2 revealed that among 20 potential bioactive peptides in alcalase-generated hydrolysate A₉, only 3 peptides, with sequences KFQWGY, SQDWSFY, and YWYPPQ, showed the highest binding toward CEase catalytic sites. Among 43 peptides in 9-h papain-generated hydrolysate P₉, 4 peptides were found to be potent CEase inhibitors. Molecular docking revealed that WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR from P₉ hydrolysates were able to bind to the active site of CEase with good docking scores and molecular mechanics–generalized born surface area binding energies. Overall, this is the first study reporting CEase inhibitory potential of peptides generated from milk proteins.     

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identi?ed from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography–electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71–77) and β-lactoglobulin 1 f(143–155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.     

乳清蛋白源生物活性肽段序列及其功能

乳清蛋白经酶解产生的肽类除了具有极高的营养价值之外．而且还具有一定的潜在生物活性。目前发现的乳清蛋白源生物活性肽主要有阿片肽、抗高血压肽、抗茵肽和免疫调节肽。综述了上述4种肽的氨基酸序列及其主要功能；对酶解过程中应注意的因素，如酶的类型、水解度、预处理方式、酶的灭活方式、底物纯度和水解过程等理化参数进行了归纳总结。     

Inhibitory properties of camel whey protein hydrolysates toward liver cancer cells,dipeptidyl peptidase-IV,and inflammation

This report describes an investigation of camel whey protein hydrolysates (CWPH) produced by gastric and pancreatic enzymes for their in vitro antidiabetic, anticancer, and anti-inflammatory properties. Degree of hydrolysis (DH) ranged from 8.54 to 47.53%, with hydrolysates generated using chymotrypsin for 6 h displaying the highest DH. Reverse phase-HPLC analysis showed that α-lactalbumin underwent complete degradation, with no intact α-lactalbumin detected in CWPH. The CWPH displayed enhanced antidiabetic activity compared with intact whey proteins; with pepsin- and chymotrypsin-generated CWPH displaying greater inhibition of dipeptidyl peptidase IV (DPP-IV), α-glucosidase, and α-amylase compared with trypsin-generated CWPH. The highest antiproliferative effect was observed for CWPH generated by chymotrypsin for 3 h, with only 4.5 to 6.5% viable liver cancer cells (HepG2) remaining when tested at concentrations from 400 to 1,000 µg/mL. The highest anti-inflammatory activity was manifested by CWPH generated by pepsin at 6-h hydrolysis. We report enhanced antiproliferative, antidiabetic, and anti-inflammatory activities upon hydrolysis of camel whey proteins, indicating their potential utilization as bioactive and functional ingredients.     

Antidiabetic,angiotensin-converting enzyme inhibitory and anti-inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic-fermented camel milk with molecular interaction study

This study was to separate and purify antidiabetic and angiotensin-converting enzyme (ACE) inhibitory peptides from Lactiplantibacillus plantarum KGL3A fermented camel milk. After 48 h of fermentation at 37°C, ɑ-amylase inhibition, ɑ-glucosidase inhibition, lipase inhibition and ACE inhibitory activities were 80.94%, 64.45%, 63.93%, and 77.53%, respectively in fermented camel milk. Optimisation of growth condition for the evaluation of maximum peptide production was evaluated by measuring proteolytic activity (O-phthalaldehyde, OPA method) with different inoculation rates and incubation times and highest proteolytic activity (9.21 mg/mL) was observed after 48 h of fermentation at 2.5% rate of inoculation. The antidiabetic and ACE inhibitory activity of 3 kDa permeate fraction were higher as compared with other fractions. Purification of antidiabetic and ACE inhibitory peptides from fermented camel milks was performed through sodium dodecyl-sulphate polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis and maximum number of protein bands were present in between 25 and 10 kDa. The generated peptide sequences were matched with antihypertensive peptide database (AHTPDB) and BIOPEP databases for confirming the antidiabetic and ACE inhibitory activity. Peptides, that is. TDVMPQWW and MMSLVSLLLVGILFPTIQAK were having highest peptide ranker score among the all sequences. Furthermore, anti-inflammatory activity (TNF-α, IL-6 and IL-1β) of fermented camel milk was evaluated in macrophage cell line RAW 264.7 (Ralph and William's cell line). Furthermore, peptides were predicted to have improved binding affinity against Human Angiotensin converting enzyme (hACE) through molecular docking.     

水牛奶乳清蛋白制备抗氧化活性肽工艺的研究

实验是以水牛奶为原料,分离纯化后得到乳清蛋白。利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶5种不同的蛋白酶对水牛奶乳清蛋白酶解以制备抗氧化活性多肽。酶筛选结果显示,中性蛋白酶是最适宜酶解水牛奶乳清蛋白制备抗氧化活性肽,其酶解液的还原能力和DPPH自由基清除率较其他4种酶高。探讨酶解反应时pH、温度、时间、酶浓度对酶解反应的水解度、酶解液的还原能力和DPPH自由基的清除率的影响,在单因素试验基础上,采用响应面法对酶解工艺进行优化。结果表明,中性蛋白酶酶解乳清蛋白的最佳工艺参数为:pH为7.4,温度为50.5℃,酶与底物浓度比为2.1%,酶解时间5.0h,此时2mg/mL酶解物的DPPH自由基清除率为32.58%。实测结果与预测值吻合效果良好。     

Invited review: Physiological properties of bioactive peptides obtained from whey proteins

Processing of whey proteins yields several bioactive peptides that can trigger physiological effects in the human body: on the nervous system via their opiate and ileum-contracting activities; on the cardiovascular system via their antithrombotic and antihypertensive activities; on the immune system via their antimicrobial and antiviral activities; and on the nutrition system via their digestibility and hypocholesterolemic effects. The specific physiological effects, as well the mechanisms by which they are achieved and the stabilities of the peptides obtained from various whey fractions during their gastrointestinal route, are specifically discussed in this review.     

In vitro antidiabetic and antihypercholesterolemic activities of camel milk protein hydrolysates derived upon simulated gastrointestinal digestion of milk from different camel breeds

Milk protein hydrolysates derived from 4 camel breeds (Pakistani, Saheli, Hozami, and Omani) were evaluated for in vitro inhibition of antidiabetic enzymatic markers (dipeptidyl peptidase IV and α-amylase) and antihypercholesterolemic enzymatic markers (pancreatic lipase and cholesterol esterase). Milk samples were subjected to in vitro simulated gastric (SGD) and gastrointestinal digestion (SGID) conditions. In comparison with intact milk proteins, the SGD-derived milk protein hydrolysates showed enhanced inhibition of α-amylase, dipeptidyl peptidase IV, pancreatic lipase, and cholesterol esterase as reflected by lower half-maximal inhibitory concentration values. Overall, milk protein hydrolysates derived from the milk of Hozami and Omani camel breeds displayed higher inhibition of different enzymatic markers compared with milk protein hydrolysates from Pakistani and Saheli breeds. In vitro SGD and SGID processes significantly increased the bioactive properties of milk from all camel breeds. Milk protein hydrolysates from different camel breeds showed significant variations for inhibition of antidiabetic and antihypercholesterolemic enzymatic markers, suggesting the importance of breed selection for production of bioactive peptides. However, further studies on identifying the peptides generated upon SGD and SGID of milk from different camel breeds are needed.     

海洋贝类蛋白源生物活性肽及肽组学的研究进展

海洋贝类作为一种原料大宗广泛易得的优质蛋白质资源,是生物活性肽开发的理想原料。本文对海洋贝类蛋白资源生物活性肽的概念、国内外研究进展进行了综述,并重点介绍了肽组学在活性肽结构鉴定中的应用,同时对贝类蛋白资源生物活性肽面临的机遇与挑战进行了分析,旨在为开发海洋贝类蛋白资源及其活性肽提供思路。      

Effect of camel milk protein hydrolysates against hyperglycemia,hyperlipidemia, and associated oxidative stress in streptozotocin (STZ)-induced diabetic rats

This study investigated the effect of camel milk protein hydrolysates (CMPH) at 100, 500 and 1,000 mg/kg of body weight (BW) for 8 wk on hyperglycemia, hyperlipidemia, and associated oxidative stress in streptozotocin-induced diabetic rats. Body weights and fasting blood glucose levels were observed after every week until 8 wk, and oral glucose tolerance test (OGTT) levels and biochemical parameters were evaluated after 8 wk in blood and serum samples. Antioxidant enzyme activity and lipid peroxidation in the liver were estimated, and histological examination of the liver and pancreatic tissues was also conducted. Results showed that CMPH at 500 mg/kg of BW [camel milk protein hydrolysate, mid-level dosage (CMPH-M)] exhibited potent hypoglycemic activity, as shown in the reduction in fasting blood glucose and OGTT levels. The hypolipidemic effect of CMPH was indicated by normalization of serum lipid levels. Significant improvement in activity of superoxide dismutase and catalase, and reduced glutathione levels were observed, along with the attenuation of malondialdehyde content in groups fed CMPH, especially CMPH-M, was observed. Decreased levels of liver function enzymes (aspartate aminotransferase and alanine aminotransferase) in the CMPH-M group was also noted. Histology of liver and pancreatic tissue displayed absence of lipid accumulation in hepatocytes and preservation of β-cells in the CMPH-M group compared with the diabetic control group. This is the first study to report anti-hyperglycemic and anti-hyperlipidemic effect of CMPH in an animal model system. This study indicates that CMPH can be suggested for its therapeutic benefits for hyperglycemia and hyperlipidemia, thus validating its use for better management of diabetes and associated comorbidities.     

鸡蛋清中功能蛋白及活性肽的研究进展

蛋清中功能蛋白及活性肽具有多种生理活性,对人类健康及疾病的预防和治疗方面的巨大潜力,成为食品领域的研究热点之一。本文综述了蛋清蛋白中的主要功能蛋白的结构及功能,生物活性肽研究进展,并对蛋清蛋白及活性肽的研究进行展望,以期为后续深入的基础研究和应用开发提供参考。      

Impact of enzyme inactivation conditions during the generation of whey protein hydrolysates on their physicochemical and bioactive properties

The thermal inactivation conditions (75 °C × 35 min, 80 °C × 10 min, 85 °C × 5 min and 90 °C × 5 min) for Protamex? following bovine whey protein concentrate (WPC) hydrolysis was studied with the view to limiting WPC hydrolysate (WPH) aggregation while maintaining bioactivity. A decrease in the amount of large WPH aggregates formed was observed at inactivation temperatures ≤85 °C. However, the WPC appeared to be more hydrolysed on heating at 75 °C × 35 min, as Protamex? was active for longer under these heating conditions. Significantly (P < 0.05), higher WPH antioxidant (oxygen radical absorbance capacity – ORAC) activity was obtained on inactivation at temperatures ≤80 °C. In contrast, the dipeptidyl peptidase IV (DPP‐IV) inhibitory properties of all WPH samples were similar (P > 0.05). A reduction in thermal treatment from 90 °C × 5 min to 85 °C × 5 min was sufficient to decrease the amount of large aggregates formed in the hydrolysate without altering its bioactive properties.     

Invited review: Dairy proteins and bioactive peptides: Modeling digestion and the intestinal barrier

Dairy products are one of the most important sources of biologically active proteins and peptides. The health-promoting functions of these peptides are related to their primary structure, which depends on the parent protein composition. A crucial issue in this field is the demonstration of a cause-effect relationship from the ingested protein form to the bioactive form in vivo. Intervention studies represent the gold standard in nutritional research; however, attention has increasingly been focused on the development of sophisticated in vitro models of digestion to elucidate the mechanism of action of dairy nutrients in a mechanistic way and significantly reduce the number of in vivo trials. On the other hand, the epithelial intestinal barrier is the first gate that actively interacts with digestion metabolites, making the intestinal cells the first target tissue of dairy nutrients and respective metabolites. An evolution of the in vitro digestion approach in the study of dairy proteins and derived bioactive compounds is the setup of combined in vitro digestion and cell culture models taking into consideration the endpoint to measure the target organism (e.g., animal, human) and the key concepts of bioaccessibility, bioavailability, and bioactivity. This review discusses the relevance and challenges of modeling digestion and the intestinal barrier, focusing on the implications for the modeling of dairy protein digestion for bioactivity evaluation.     

Molecular basis of the anti-diabetic properties of camel milk through profiling of its bioactive peptides on dipeptidyl peptidase IV (DPP-IV) and insulin receptor activity

The molecular basis of the anti-diabetic properties of camel milk reported in many studies and the exact active agent are still elusive. Recent studies have reported effects of camel whey proteins (CWP) and their hydrolysates (CWPH) on the activities of dipeptidyl peptidase IV (DPP-IV) and the human insulin receptor (hIR). In this study, CWPH were generated, screened for DPP-IV binding in silico and inhibitory activity in vitro, and processed for peptide identification. Furthermore, pharmacological action of intact CWP and their selected hydrolysates on hIR activity and signaling and on glucose uptake were investigated in cell lines. Results showed inhibition of DPP-IV by CWP and CWPH and their positive action on hIR activation and glucose uptake. Interestingly, the combination of CWP or CWPH with insulin revealed a positive allosteric modulation of hIR that was drastically reduced by the competitive hIR antagonist. Our data reveal for the first time the profiling and pharmacological actions of CWP and their derived peptides fractions on hIR and their pathways involved in glucose homeostasis. This sheds more light on the anti-diabetic properties of camel milk by providing the molecular basis for the potential use of camel milk in the management of diabetes.     

Application and bioactive properties of proteins and peptides derived from hen eggs: opportunities and challenges

Several proteins and peptides that are released in vitro and/or in vivo from hen eggs are biologically active and have a variety of functional properties in humans beyond normal nutrition, for which extensive studies have been performed. This review focuses on their biological activities, including antihypertensive, antioxidant, antimicrobial, antiadhesive, immunomodulatory and antithrombotic activities and enhancement of mineral absorption. These proteins and peptides have been shown to regulate the nervous system, cardiovascular system, immune system and gastrointestinal system. The potential application and future directions of research on these bioactive peptides and proteins in the food industry are also addressed. © 2014 Society of Chemical Industry     

The potential application of the protein hydrolysates of three medicinal plants: cytotoxicity and functional properties

Functional evaluation of encrypted bioactive peptides in protein structure helps to better understand those for using in pharmacy and food sciences. For this purpose, the total protein was extracted from Matricaria chamomilla, Ziziphora clinopodioides, and Cressa cretica, and partially purified with ammonium sulfate. Protein hydrolysates were obtained from pancreatin hydrolysis for 240 min and the enzyme hydrolysis was confirmed using the determination of hydrolysis degree and Fourier transform infrared (FT-IR) followed by the physicochemical and sensory properties were investigated. The results showed that all hydrolysates had both cytotoxic and antioxidant activities. Specifically, C. cretica hydrolysates represented cytotoxic activity against the MCF-7 cell line with the IC₅₀ of 135.21 µg/mL, while showed no significant growth inhibition effect on the HEK293 cell line. Besides, M. chamomilla hydrolysates showed the lowest bitterness value (1.125 ± 0.52). From the perspective of color investigation, M. chamomilla hydrolysates indicated the highest L^* and the lowest a^* factors. The highest turbidity and surface tension, and 10-fold more cancer cell killing effect under gastrointestinal digestion conditions were observed for M. chamomilla hydrolysates. Therefore, bioactive peptides might be formulated in designing of novel anticancer drugs or could be used in promising protocols for the production of food products with beneficial health effects.     

Comparison of bioactive peptides prepared from sheep cheese whey using a food‐grade bacterial and a fungal protease preparation

Novel bacterial (HT) and fungal (FPII) food‐grade protease preparations were evaluated for their ability to hydrolyse sheep cheese whey (SCW) and the generation of bioactive peptides. Both protease preparations hydrolysed the whey proteins to small peptides over 24‐h hydrolysis time, but the time course hydrolysis profiles were different as evaluated by SDS‐PAGE. The HT whey hydrolysate had considerably higher antioxidant and angiotensin‐I converting enzyme (ACE)‐inhibitor activity than the FPII hydrolysate. Neither hydrolysate was cytotoxic towards Vero cells. OFFGEL electrophoresis of the small peptide pool fraction (<15 amino acids) of each hydrolysate indicated differences in the pI distribution of the bioactive peptides. This likely reflects the diverse hydrolytic specificity of the proteases. Although the antioxidant activity of both hydrolysates was not significantly affected by simulated gastrointestinal digestion, the loss of ACE‐inhibitor activity was greater with the FPII hydrolysate.     

Identification and molecular mechanisms of novel antioxidative peptides from fermented camel milk (Kachchi breed,India) with anti-inflammatory activity in raw macrophages cell lines

The investigation was aimed at assessing anti-inflammatory and antioxidative activities along with the release of peptides with antioxidative properties during the fermentation of camel milk by Lacticaseibacillus casei (NK9). Reverse-phase high-performance liquid chromatography (RP-HPLC) was used to separate the bioactive peptides of 3 and 10 kDa (permeates and retentates). Reverse-phase liquid chromatography–mass spectrometry (RPLC/MS) was used to identify and characterise the pure bioactive peptides, and the effect of fermented camel milk on inflammation produced by lipopolysaccharide (LPS)/endotoxin in RAW 264.7 (Ralph and William's cell line) was also examined. Furthermore, docking revealed that peptides (LLNEK and IYTFPQPQSL) were predicted to inhibit myeloperoxidase (nMPO) activity by engaging with different residues in and around the human myeloperoxidase (hMPO) active site.     

Identification and characterization of novel angiotensin-converting enzyme inhibitors obtained from goat milk

The aim of the present study was to identify and characterize new inhibitory peptides of angiotensin I-converting enzyme (ACE) from goat milk and to analyze the effect of long-term intake of a goat milk hydrolysate-supplemented (GP-hyd) diet on the development of hypertension in spontaneously hypertensive rats (SHR). Three new inhibitory peptides for ACE (TGPIPN, SLPQ, and SQPK) were isolated. The inhibitory concentration 50% (IC₅₀) values of individual peptides were 316, 330, and 354 μmol/L, respectively. Only TGPIPN was found to pass intact a monolayer of Caco-2 cells in small amounts. The SHR fed for 12 wk a diet (GP-hyd) enriched in a hydrolysate containing these peptides (estimated intake of TGPIPN was 230 mg/kg per d) showed lower (approximately 15 mmHg) systolic blood pressure than animals fed a control diet. The ACE activities in the aorta, left ventricle, and kidney were significantly decreased in the GP-hyd group compared with those of the control group and were similar to those found in SHR fed captopril (130 mg/kg per d). Impaired endothelium-dependent relaxation to acetylcholine by aortic rings from SHR was improved in those fed the GP-hyd diet. The left ventricle weight and kidney weight index were significantly reduced in the GP-hyd group and captopril groups. Moreover, long-term treatment of SHR with a diet enriched in goat milk hydrolysate, or captopril, attenuated the development of hypertension, cardiac and renal hypertrophy, and endothelial dysfunction. These effects might be related to the in vivo inhibitory effects of the hydrolysate on tissue ACE activity.     

Preparation and analysis of hydrolysates from whey protein concentrate using the proteases from Bacillus licheniformis and Aspergillus oryzae

The action of proteases from Bacillus licheniformis and Aspergillus oryzae was studied with the aim of preparing hydrolysates from whey protein concentrate with nutritionally appropriate peptide profile. Various enzyme/substrate ratios were used, and the peptides were fractionated by size‐exclusion HPLC followed by their quantification using the rapid method of correct fraction area (CFA). The protease from B. licheniformis (E:S of 8:100) produced the best peptide with a much lower amount of large peptides (44.61%), greater of di‐ and tripeptides (8.79%) and higher sum of the di‐ and tripeptides with free amino acids (9.99%) than the other hydrolysates. The advantage of using a lower E:S ratio to obtain a nutritionally adequate peptide profile was observed for the protease of A. oryzae when it passed from 3:100 to 2:100.