The fasting hypoglycemic effect of casein hydrolysate (CH) was investigated in db/db diabetic-like mice using a multiomics integrated analysis of peptidome, transcriptome, and metabolome. Results showed that the oral administration of CH at a dose of 600 mg/kg/day for 4 weeks reduced the fasting blood glucose levels by 14.73 ± 9.77%, alleviated insulin resistance (HOMA-IR index) by 36.91 ± 22.62%, and mitigated hepatic damage in db/db diabetic-like mice. Hepatic differential metabolites after CH treatment were enriched in Glu-related metabolites, which acted as substrates for the TCA cycle, enhancing hepatic glucose consumption. The hepatic transcriptomic results revealed that CH treatment upregulated (p < 0.05) hub gene expressions of pparg and pik3cb, leading to an activation of the PPAR signaling pathway, further improving the insulin/PI3K/AKT signaling pathway. The hub gene expressions were highly correlated with Glu-related metabolites in multiomics integrated analysis. Glx/Glx-containing peptides (Glx represents Glu and Gln) in CH, as a dietary supplement to increase hepatic Glu-related metabolites, might be the key active component responsible for its hypoglycemic effect. Particularly, the supplement of Glx was confirmed to effectively (p < 0.05) enhance glucose consumption in hepatocytes. This provides a basis for the development of CHs as functional food.

Background: Due to the unique structure of proline (Pro), Pro-containing peptides (PCPs) are endowed with specific conformational and physiological properties. There has been an increasing interest in the health- promoting effects of PCPs from dietary proteins. However, a comprehensive overview of the bioactivities, preparation, and bioavailability of food-derived PCPs is lacking. Scope and approach: This article provides a comprehensive discussion of recent advances in food sources, available production methods, structure-activity relationships underlying the bioactivities, and bioavailability of food-derived PCPs. Future research directions are also discussed in optimizing PCPs as health-beneficial functional food ingredients. Key findings and conclusions: Food-derived PCPs exhibit diverse bioactivities highlighting their potential human health benefits, including ACE inhibition, antioxidant effects, DPP-IV inhibition, and opioid-like effects. The role of Pro, protein source preferences, and production methods for each bioactivity are distinct. Pro-specific peptidases from the gastrointestinal tract, intestinal epithelial cells, and blood are responsible for the degradation of PCPs in vivo. Further investigation is needed to develop PCPs-specific production methods, improve the bioavailability of PCPs, and validate their bioactivity in clinical trials.

This study investigated the metabolic stability of the casein-derived peptide IPIQY and its active fragments in relation to in vivo hypoglycemic activity. IPIQY dose dependently inhibited DPP-IV activity in Caco-2 cells (IC50 = 73.48 μM). When administered at 300 μmol/kg, IPIQY reduced intestinal and plasma DPP-IV activity by 46.46% and 13.75%, respectively, enhanced plasma insulin levels by 43.42%, and reduced blood glucose levels by 37.77% in OGTT in mice. Metabolic stability results showed IPIQY rapidly entered the bloodstream reaching a peak plasma concentration of 0.51 μM at 2 min after oral administration (300 μmol/kg). It was stable in gastric digestion but degraded into IPIQ and IPI in the intestine. Its low permeability ((4.40 ± 0.18) × 10-8 cm/s) across Caco-2 cells was mainly due to degradation into IP by BBM. IPI was identified as the most active metabolized fragment. The peptides IPIQY, IPIQ, and IPI, all containing active fragment IPI, exhibited comparable hypoglycemic effects in vivo, whereas IP was significantly less active. Furthermore, the gastrointestinal degradation of IPIQY releases active IPI, contributing to its superior hypoglycemic effect via oral administration compared to intravenous injection. These findings provide new insights into the hypoglycemic potential and administration routes of IPIQY.

BACKGROUND: Casein hydrolysates have attracted much interest as anti-diabetic food, but their hypoglycemic mechanism and biopeptides are not well understood. This study aimed to explore the anti-diabetic mechanism and potential biopeptides of casein hydrolysates in streptozotocin/high-fat-diet-induced diabetic rats and HepG2 cells.RESULTS: Oral administration of casein hydrolysate prepared with papain-Flavourzyme combination (P-FCH) decreased fasting blood glucose, improved oral glucose tolerance, and reduced HbA1c values in diabetic rats. P-FCH was ineffective in alleviating insulin resistance (homeostasis model assessment and insulin sensitivity index) and enhancing hepatic insulin signaling transduction (phosphorylated Akt, hexokinase activity, and pyruvate kinase activity) in diabetic rats. However, P-FCH significantly upregulated adenosine monophosphate-activated protein kinase phosphorylation and glucose transporter-2 expression, inhibited phosphoenolpyruvate carboxylase kinase activity, and elevated glycogen content in liver tissue of diabetic rats. Furthermore, P-FCH increased glucose consumption independently in normal and insulin-resistant HepG2 cells without the presence of insulin. The peptide composition of P-FCH was characterized. The potential biopeptides in P-FCH showed the sequence characteristic of a Val at the N-terminal or a Pro at the P2 position, and the hypoglycemic activity of Val-Pro-Leu-Gly (the most potential biopeptide in P-FCH) was verified by oral glucose tolerance test in mice.CONCLUSION: These results suggested that activation of the non-insulin-mediated AMPK pathway might be the determinant mechanism of P-FCH on the hypoglycemic effect. The novel peptide Val-Pro-Leu-Gly in P-FCH was effective in reducing blood glucose levels when orally administered to mice.(c) 2023 Society of Chemical Industry.

This study focused on the enzymatic characteristics, with regard to primary specificity, secondary specificity, and hydrolysis ability of five proteases (Alcalase, Protamex, papain, Flavourzyme, and ProtexA), towards the release of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from bovine casein. The secondary specificity of the five proteases, especially the release capability for Xaa-Pro-type peptide played a determining role in the release of DPP-IV inhibitory peptides from bovine casein. Moreover, protease combinations, chosen based on their Xaa-Pro-type peptide release abilities, resulted in superior DPP-IV inhibitory activity compared to other combinations. Particularly, the hydrolysate with the highest DPP-IV inhibitory activity (IC50 value of 0.45 ± 0.07 mg/mL) was obtained by the combination hydrolysis of ProtexA and papain, achieving a 74.63% ± 1.62% (peak area) release of Xaa-Pro-type peptide, and a significant improving glucose tolerance in mice (33.42% ± 1.68% reduction of blood glucose AUC compared with control mice). The mechanistic insights revealed that papain efficiently released long Xaa-Pro-type peptides in the early hydrolysis stage, and subsequently, the synergistic effect of papain and ProtexA led to an accelerated and extensive release of short Xaa-Pro-type peptides. These findings provide valuable insight into enzymatic processes for generating DPP-IV inhibiting peptides from casein.

Phage therapy has the potential to alleviate plant bacterial wilt. However, the knowledge gap concerning the phage-agrochemical interaction impedes the broader application of phages in agriculture. This study characterized a phage isolate and investigated its interactions with agrochemicals. A novel species within the Ampunavirus genus was proposed, serving phage LPRS20 as a type phage with a broad lytic range and significant antibacterial activity against Ralstonia solanacearum strains infecting tobacco, chili, or tomato. Sensory evaluation of the morphology of tobacco leaves suggested that phage application resulted in negligible harm to plants. Investigations into phage-agrochemical interactions revealed synergisms when LPRS20 was delivered 4 h before thiodiazole-copper as well as LPRS20 in combination with low-concentration berberine. Overall, our findings reveal that phage LPRS20 represents a novel, effective, and eco-friendly biocontrol agent against tobacco bacterial wilt in vivo and in vitro and contributes to the potential integration of phages and agrochemicals for controlling soil-borne pathogens.

Dipeptidyl peptidase-IV (DPP-IV) inhibiting peptides have attracted increased attention because of their possible beneficial effects on glycemic homeostasis. However, the structural basis underpinning their activities has not been well understood. This study combined computational and in vitro investigations to explore the structural basis of DPP-IV inhibitory peptides. We first superimposed the Xaa-Pro-type peptide-like structures from several crystal structures of DPP-IV ligand-protein complexes to analyze the recognition interactions of DPP-IV to peptides. Thereafter, a small set of Xaa-Pro-type peptides was designed to explore the effect of key interactions on inhibitory activity. The intramolecular interaction of Xaa-Pro-type peptides at the first and third positions from the N-terminus was pivotal to their inhibitory activities. Residue interactions between DPP-IV and residues of the peptides at the fourth and fifth positions of the N-terminus contributed significantly to the inhibitory effect of Xaa-Pro-type tetrapeptides and pentapeptides. Based on the interaction descriptors, quantitative structure-activity relationship (QSAR) studies with the DPP-IV inhibitory peptides resulted in valid models with high R-2 values (0.90 for tripeptides; 0.91 for tetrapeptides and pentapeptides) and Q(2) values (0.33 for tripeptides; 0.68 for tetrapeptides and pentapeptides). Taken together, the structural information on DPP-IV and peptides in this study facilitated the development of novel DPP-IV inhibitory peptides.

Enzymatic hydrolysis can not only increase the digestibility of casein, but also cause bitterness. This study aimed to investigate the effect of hydrolysis on the digestibility and bitterness of casein hydrolysates and provided a novel strategy for the preparation of high-digestibility and low-bitterness casein hydrolysates based on the release pattern of bitter peptides. Results showed that with the increase of the degree of hydrolysis (DH), the digestibility and bitterness of hydrolysates increased. However, the bitterness of casein trypsin hydrolysates rapidly increased in the low DH range (3%-8%), while the bitterness of casein alcalase hydrolysates rapidly increased in a higher DH range (10.5%-13%), indicating the discrepancy in the release pattern of bitter peptides. Peptidomics and random forests revealed that peptides containing >6 residues with hydrophobic amino acids (HAAs) at the N-terminal and basic amino acids (BAAs) at the C-terminal (HAA-BAA type) obtained from trypsin contributed more to the bitterness of casein hydrolysates than those containing 2-6 residues. On the other hand, peptides containing 2-6 residues with HAAs at both N- and C-terminals (HAA-HAA type) released by alcalase contributed more to the bitterness of casein hydrolysates than those containing >6 residues. Furthermore, a casein hydrolysate with a significantly lower bitter value containing short-chain HAA-BAA type peptides and long-chain HAA-HAA type peptides from the combination of trypsin and alcalase was obtained. The digestibility of the resultant hydrolysate was 79.19% (52.09% higher than casein). This work is of great significance for the preparation of high-digestibility and low-bitterness casein hydrolysates.

This study revealed the effect of hydrolysis on the digestibility and bitterness of casein hydrolysates (CHs) and provided a novel strategy for the preparation of high-digestibility and low-bitterness CHs. Results showed that both the enzyme and degree of hydrolysis (DH) affected the digestibility and bitterness, while trypsin, alcalase and flavourzyme were more suitable to prepare target CHs. Peptidomics and random forests revealed that for bitter peptides with hydrophobic amino acids (HBAA) at N-terminal and basic amino acids (BAA) at C-terminal (HBAA-BAA type) released by trypsin, the long-chain ones contributed bitterness more than the short-chain ones. For bitter peptides with HBAA at both N-terminal and C-terminal (HBAA-HBAA type) released by alcalase, the short-chain ones contributed bitterness more than the long-chain ones. Furthermore, a low-bitterness hydrolysate contained shorter HBAA-BAA type peptides and longer HBAA-HBAA type peptides was provided. This work is of guiding significance for the preparation of high-digestibility and low-bitterness CHs.

The objective of this study was to explore the molecular targets and mechanism of Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) on regulating glucose metabolism in hepatic cells and their in vivo hypoglycemic activities in mice. Results showed that both IPP and VPP (600 μM) significantly enhanced the glucose consumption in HepG2 cells and primary hepatocytes (p < 0.05). They also regulated activities of glucose metabolizing enzymes and increased the protein expression of p-AKT and GLUT2 in HepG2 cells. IPP directly interacted with the insulin receptor (IR) to activate the insulin/AKT signaling pathway. The activity of VPP on glucose consumption was not attributed to IR binding, and 76 potential antidiabetic targets were predicted by similarity ensemble and shape similarity approaches. Among them, the AKT and MAPK signaling pathway, in which two hub genes AKT1 and MAPK4 existed, were evaluated to make major contributions to the activity of VPP on glucose consumption. Moreover, both IPP and VPP (300 μmol/kg) could significantly reduce the blood glucose levels in mice (p < 0.05), with blood glucose area under the curve dropping by approximately 19% ± 0.09 and 21% ± 0.11%, respectively. This study provides a new theoretical support for the development of IPP and VPP as functional foods to regulate glucose metabolic disorders.

通过抑制二肽基肽酶Ⅳ(Dipeptidyl-peptidase Ⅳ, DPP-Ⅳ)的活性,从而减少GLP-1的降解,提高血液中胰岛素的水平,是控制血糖水平的一种重要手段.本文以海参(Holotharia tubulosa)为原料,通过探究蛋白酶种类、加酶量和酶解时间对酶解产物DPP-Ⅳ抑制活性、蛋白回收率和水解度的影响,确定了海参DPP-Ⅳ抑制肽的制备条件,并进一步测定了其分子量分布和总氨基酸组成,最后通过UPLC-MS/MS鉴定了其潜在的活性肽序列.结果 发现,木瓜蛋白酶与复合蛋白酶1∶1的复配酶解具有最佳的酶解效果,其产物的得率和DPP-Ⅳ抑制活性均最高,且在加酶量为干海参质量的1％,酶解时间为4h时,海参酶解产物在终浓度为2mg/mL时的DPP-Ⅳ抑制率为66.97％,蛋白回收率为76.25％,水解度为6.10％.酶解产物的分子量大都小于5000u,且富含脯氨酸(Pro)和丙氨酸(Ala)等与抑制DPP-Ⅳ活性有关的氨基酸.将获得的酶解物中的肽段进行液质联用检测,并通过Mascot分析筛选得到28条具DPP-Ⅳ抑制肽的肽序列,分子量在500～1936u.本实验结果为以海参为原料进行降血糖产品的开发奠定了基础.

Hyperglycemia-induced oxidative stress can cause liver damage in diabetes, and protein hydrolysates with antidiabetic and antioxidant properties are emerging as a potential therapy. In this study, protective effects of casein hydrolysates against live oxidative damage in streptozotocin/high-fat-induced diabetic rats were studied and potentially bioactive peptides were explored by an integrated approach of differential peptide and in silico analysis. Results showed that different casein hydrolysates significantly alleviated liver oxidative damage (p < 0.05) via different mechanisms. Particularly, casein hydrolyzed by a papain-flavourzyme combination (P-FCH) treatment significantly improved liver antioxidant enzyme activities by enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) transcription (p < 0.05). Furthermore, 18 peptides were screened as potential bioactive peptides by analyzing differential peptides among different hydrolysates combined with in silico prediction. Among them, the dipeptide WM might directly inhibit the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 interaction as potential Nrf2 activators. These results suggested that P-FCH might be an alternative way to treat liver damage in diabetes.

本文为了探讨酪蛋白源七肽AVPYPQR在不同体系中的抗氧化活性及构效关系,比较研究了AVPYPQR及其相关片段PYPQ、PYPQR、vPYPQ、VPYPQR和AVPYPQ在体外化学方法和细胞模型中的抗氧化能力.结果 表明,在ABTS自由基清除能力和氧自由基吸收能力(ORAC)等体外化学方法中6条肽的活性都高于标准抗氧化剂Trolox,其中Val、Ala和Arg残基的同时存在降低AVPYPQR的ABTS自由基清除能力,而Arg残基存在提升了AVPYPQR的ORAC活性.在细胞氧化损伤模型中,6条多肽对AAPH诱导血红细胞溶血都有保护作用,其中AVPYPQR保护效果最好,Ala和Arg残基存在提升其保护效果;H2O2诱导HepG2细胞氧化损伤模型中,除PYPQ,其他多肽都具有显著保护效果(p＜0.05),其中Ala、Vai和Arg残基都可以提升保护效果,但是没有叠加作用.综上,除PYPQ,其它5条多肽在在体外化学方法和细胞模型中都表现出较强抗氧化活性,但无显著性相关,且不同方法中Val、Ala和Arg残基对AVPYPQR活性影响不同.