Casein Peptides Research Articles

Functional Peptides from Yak Milk Casein: Biological Activities and Structural Characteristics.

The average content of casein in yak milk is 40.2 g/L. Casein can be degraded by enzymatic digestion or food processing to produce abundant degradation peptides. International researchers have studied the degradation peptides of yak milk casein by using multiple techniques and methods, such as in vitro activity tests, cellular experiments, proteomics, bioinformatics, etc., and found that the degradation peptides have a wide range of functional activities that are beneficial to the human body, such as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, anti-inflammatory, antidiabetic, antimicrobial, anticancer, and immunomodulatory activities, etc., and it has been proved that the types and strengths of functional activities are closely related to the structural characteristics of the peptides. This paper describes the characteristics of yak milk proteins, the functional activities, and mechanism of action of degraded peptides. Based on the types of functional activities of yak milk casein degradation peptides, we classified and elucidated the effects of structural factors, such as peptide molecular weight, peptide length, amino acid sequence, physicochemical properties, electrical charge, hydrophobicity, spatial conformation, chain length, and the type of enzyme on these activities. It reveals the great potential of yak milk casein degradation peptides as functional active peptide resources and as auxiliary treatments for diseases. It also provides important insights for analyzing yak casein degradation peptide activity and exploring high-value utilization.

Exploring structural features of sleep-enhancing peptides derived from casein hydrolysates by chemometrics and random forest methodology

Milk casein is regarded as source to release potential sleep-enhancing peptides. Although various casein hydrolysates exhibited sleep-enhancing activity, the underlying reason remains unclear. This study firstly revealed the structural features of potential sleep-enhancing peptides from casein hydrolysates analyzed through peptidomics and multivariate analysis. Additionally, a random forest model and a potential Tyr-based peptide library were established, and then those peptides were quantified to facilitate rapidly-screening. Our findings indicated that YP-, YI/L, and YQ-type peptides with 4–10 amino acids contributed more to higher sleep-enhancing activity of casein hydrolysates, due to their crucial structural features and abundant numbers. Furthermore, three novel strong sleep-enhancing peptides, YQKFPQY, YPFPGPIPN, and YIPIQY were screened, and their activities were validated in vivo. Molecular docking results elucidated the importance of the YP/I/L/Q- structure at the N-terminus of casein peptides in forming crucial hydrogen bond and π-alkyl interactions with His-102 and Asn-60, respectively in the GABAA receptor for activation.

Effect of casein bioactive peptides on performance, nutrient digestibility, enzyme activity and intestinal microbial population in broiler chickens.

This experiment was carried out to investigate the effect of bioactive peptides derived from casein on performance, nutrient digestibility, enzyme activity and intestinal microbial population in broiler chickens. In this study, 350 1-day-old male Ross 308 broiler chicks were distributed among 35 pens in a completely randomized design with seven treatments, five replicates and 10 chicks in each replicate. The experimental treatments included: basal diet without any additives (control), basal diet + Avilamycin antibiotic, basal diet + 200, 400, 600, 800 and 1000 mg of peptides per kg of diet. Results showed no significant effects of the experimental treatments on weight gain and feed conversion ratio during the starter period, but there was a significant improvement in weight gain in grower, finisher and whole periods in chicks fed with diet containing 1000 mg/kg peptides (p < 0.05). Adding peptides improved intestinal morphology (in duodenum and ileum) (p < 0.05). Supplementation of casein peptides significantly reduced thiobarbituric acid reactive substances concentration in breast and thigh meat. The activity of amylase, lipase and protease enzymes improved in treatments containing 800 and 1000 mg peptides in comparison to the control treatment. The use of casein-peptides increased intestinal Lactobacillus and decreased Coliform populations.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Comparative detection of goat's milk casein through various enzyme‐linked immunosorbent assay (ELISA) approaches using caprine alpha‐S1 casein peptides and bovine casein polyclonal antibodies

SummaryCasein causes allergic reactions in cow's milk protein allergy (CMPA) individuals. Casein can cross‐react with cows and goats. Hence, a rapid and highly specific technique is required for its detection. The study aims to evaluate and optimise multiple Enzyme‐Linked Immunosorbent Assay (ELISA) approaches for the detection of casein isolated from goat's milk. The study utilised and optimised various ELISA approaches, including direct, indirect and sandwich with various antibodies. Several parameters of ELISA including pre‐treatments of casein, blocking reagent, concentrations of antibodies and background signal were evaluated. The results showed that sandwich ELISA revealed a good signal‐to‐noise ratio at 1 mg mL−1. Indirect ELISA showed a higher signal‐to‐noise ratio without any heat treatment in comparison to the direct ELISA despite utilising an equivalent concentration (i.e., 10 μg mL−1) of goat's milk casein. The findings provide a theoretical foundation for obtaining a high signal‐to‐noise ratio for antigenicity studies of goat's milk casein to minimise its immunological response.

Reuse of waste casein peptides to capture Cu (II) for long-term antibacterial reutilization

To date, researchers have worked on developing new pathways to control and manage food and industrial wastes (e.g. dairy wastewater, heavy metals, etc.), but most do not involve the reuse of these wastes. Herein, we propose to use casein peptides (CPs) based on dairy waste to capture Cu2+ from industrial wastewater, which is reduced to copper and cuprous oxide nanoparticles (consisting of metallic Cu and Cu2O crystalline phases) distributed on CPs. The prepared product CPs/Cu/Cu2O NPs showed prominent antibacterial activity against Escherichia coli and Staphylococcus aureus, as well as multidrug-resistant bacteria from livestock wastewater through generating reactive oxygen species (ROS) in combination with released Cu2+. The method proposed in this study is also applicable to extract Cu2+ from actual electroplating wastewater for bacterial disinfection. Finally, we achieved the modulation of the composition of Cu/Cu2O NPs loaded on CPs.

Potential Prebiotic Properties of Whey Protein and Glycomacropeptide in Gut Microbiome.

Proteins in whey have prebiotic and antimicrobial properties. Whey protein comprises numerous bioactive proteins and peptides, including glycomacropeptide (GMP), a hydrophilic casein peptide that separates with the whey fraction during cheese making. GMP has traditionally been used as a protein source for individuals with phenylketonuria and also has prebiotic (supporting the growth of Bifidobacterium and lactic acid bacteria) and antimicrobial activities. GMP supplementation may help positively modulate the gut microbiome, help treat dysbiosis-related gastrointestinal disorders and improve overall health in consumers.

Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, β-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Combination of Walnut Peptide and Casein Peptide alleviates anxiety and improves memory in anxiety mices.

Anxiety disorders continue to prevail as the most prevalent cluster of mental disorders following the COVID-19 pandemic, exhibiting substantial detrimental effects on individuals' overall well-being and functioning. Even after a search spanning over a decade for novel anxiolytic compounds, none have been approved, resulting in the current anxiolytic medications being effective only for a specific subset of patients. Consequently, researchers are investigating everyday nutrients as potential alternatives to conventional medicines. Our prior study analyzed the antianxiety and memory-enhancing properties of the combination of Walnut Peptide (WP) and Casein Peptide (CP) in zebrafish. Based on this work, our current research further validates their effects in mice models exhibiting elevated anxiety levels through a combination of gavage oral administration. Our results demonstrated that at 170 + 300 mg human dose, the WP + CP combination significantly improved performances in relevant behavioral assessments related to anxiety and memory. Furthermore, our analysis revealed that the combination restores neurotransmitter dysfunction observed while monitoring Serotonin, gamma-aminobutyric acid (GABA), dopamine (DA), and acetylcholine (ACh) levels. This supplementation also elevated the expression of brain-derived neurotrophic factor mRNA, indicating protective effects against the neurological stresses of anxiety. Additionally, there were strong correlations among behavioral indicators, BDNF (brain-derived neurotrophic factor), and numerous neurotransmitters. Hence, our findings propose that the WP + CP combination holds promise as a treatment for anxiety disorder. Besides, supplementary applications are feasible when produced as powdered dietary supplements or added to common foods like powder, yogurt, or milk.

Peptidomics approaches to the discovery and ACE inhibitory effect of casein peptides derived from fermented bovine milk by kefir grains

IntroductionKefir grains with efficient proteolytic system is an excellent starter culture for the production of bioactive peptides and milk products. This study explores the casein peptides derived from fermented bovine milk by kefir grains using the peptidomics approaches. The angiotensin converting enzyme (ACE) inhibitory activity of these peptides were also investigated.MethodsAfter fermentation, peptidomics based on the LC-MS/MS was used to investigate the dynamic profile and the structure specificity of generated peptides. The ACE inhibitory activity of peptides was determined by measuring the amount of hippuric acid (HA) by a spectrophotometer at 228 nm.ResultsThe results indicated that the cell envelope proteinases (CEPs) were the PI-/PIII-type. A total of 122 peptides were identified. The β-casein was preferentially hydrolyzed by kefir grains, and the main hydrolysis regions were f57-93, f132-160 and f192-209. The αs1-, and κ-casein were also hydrolyzed by a weaker degree. In the process of fermentation, the accumulated peptides increased with the fermentation time. The fermentation products exhibited ACE inhibitory activity, and this bioactivity remained 63% after simulated gastrointestinal (GI) digestion in vitro. Additionally, 14 Pro-containing peptides with ACE inhibitory activity were also identified.ConclusionThese results provide new insights and evidence to investigate the bioactive milk peptides generated by kefir grains fermentation, as well as a reference for the development of functional foods.

Absorption characteristics and the effect on vascular endothelial cell permeability of an anticoagulant peptide

In the former report, the casein peptide TKLTEEEKNR (PfCN) exhibits strong thrombin inhibitory activity in vitro. Its absorption capabilities, however, are unclear. Therefore, we studied its absorption characteristics both in vivo and in vitro. PfCN was carried by cells from the apical chamber to the basolateral chamber via active translocation in Caco-2 cells. Meanwhile, it can also be transported by HUVECs. We found that PfCN can be taken up by HUVECs using confocal laser imaging. PfCN has been proven to have good absorption properties in in vivo experiments. After five minutes of oral treatment, PfCN was identified in the blood, peaking at 82.75 ± 36.52 ng/mL in 30 min. And PfCN vanished from the blood circulation after 120 min. According to in vivo experiments, excessive concentrations of PfCN will alter the permeability of HUVECs. As a result, there is a foundation for PfCN application in the food sector. Meanwhile, we also hope this article can give an idea to the researchers who studying the absorption of functional peptides.

Safe food through better labelling; a robust method for the rapid determination of caprine and bovine milk allergens.

Accidental milk cross-contamination is one of the most common causes for costly food recalls. Yet, quantifying trace-levels of allergen is time-consuming and current methods are not adapted for routine analyses making quality control for trace-level allergen content impractical. This perpetuates voluntary "may-contain" statements that are unhelpful for people suffering from food allergies. Here, we developed a rapid LC-MS method enabling milk allergen quantification by comparing all tryptic-peptides of major milk allergens. The bovine-specific αS-2 casein peptide and allergen-epitope NAVPITPTLNR provided excellent performance in sensitivity (LOD 1mg.kg-1; LOQ 2mg.kg-1) across various dairy products, good recovery rates in baked croissants (77% with a 10% inter-day RSD) and a linear range of 2-2,000mg.kg-1. The method can be used for routine determination of trace-contamination with bovine milk allergen and the adulteration of high-value caprine dairy products with lower-value bovine milk products, protecting consumer trust and the growing population suffering from food allergies.

Effects of different food ingredients and additives on the digestibility of extruded and roller-dried maize starch and its application in low glycemic index nutritional formula powder.

Complex interactions among starch, protein, and fat occurred during food processing affects the taste, texture, and digestibility of starch-based food. The physicochemical properties of starch, in particular the slow digestion property, are greatly influenced by the processing techniques such as extrusion and roller-drying treatment. This study argued the effects of various food ingredients and additives on the digestion properties of maize starch treated with extrusion and roller-drying, and designed a nutritional formula for developing low glycemic index products. The extruded group containing raw maize starch, soybean protein isolate, soybean oil, lecithin and microcrystalline cellulose in the ratio of 580:250:58:20:3 had the best slow digestion property. Nutritional formulas were designed at the above ratio, with the supplementary materials including calcium casein peptide, multi-vitamins, sodium ascorbate, fructooligosaccharides, xylitol and peanut meals. The sample containing 10% peanut meal and a 1:3 ratio of fructooligosaccharides and xylitol additions obtained the highest scores in sensory evaluation. A significant slow digestion effect was observed in samples produced from the optimal formula. The results of present study could provide a specific formula and may contribute to the development and production of the low glycemic index nutritional powder. This article is protected by copyright. All rights reserved.

Identification of Characteristic Peptides of Casein in Cow Milk Based on MALDI-TOF MS for Direct Adulteration Detection of Goat Milk

It is widely acknowledged that casein is an important allergenic protein in milk which may cause danger to customers. The identification and confirmation of caseins through mass spectrometry requires the selection of suitable characteristic peptides. In this study, by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the three most representative specific peptides of caseins in cow milk were screened out with mass-to-charge ratios (m/z) of 830, 1195, and 1759, respectively. By comparing 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) MALDI matrices, it was found that DHB was more suitable for peptide detection with the limits of detection (LODs) of 0.1 mg/L for α, β-casein. Furthermore, on the basis of verifying the characteristic peptides of casein from cow milk, this protocol was applied to goat milk authentication. Cow milk addition in goat milk was investigated by using the screened specific peptides. The results showed that the adulteration could be identified when the proportion of cow milk was 1% or more. When applied to inspect adulteration in five brands of commercial goat milk, specific peptides of bovine casein were detected in four of them. The method has the advantages of strong reliability, high throughput, simple preprocessing, and fast speed, which can provide powerful help for prewarning dairy allergen.

Goat casein peptides and their potential through an &lt;i&gt;in silico&lt;/i&gt; approach

The caprine caseins αs1, αs2, β, and κ from UniProtKB were characterised for amino acid profile, and hydrolysed in silico with pepsin or trypsin. The generated peptides were characterised for physical-defined properties, bioactive potential, Boman index, toxicity and allergenicity. The peptides generated are bioactive, predominating dipeptidyl peptidase IV and ACE inhibitors activities. Only two peptides are toxic, and most have been proposed to be allergenic by Allergen Online or SDAP, because they have < 50% identity with other confirmed allergenic proteins, including bovine milk caseins, however, they were considered non-allergenic by the methods MEME/MAST and BLAST in Algpred, requiring more studies for better evaluation. Despite of variations, bioinformatics tools are useful before bench tests, because allows time and cost savings, and provides studies projection for use by pharmaceutical and food industries, besides to identify sequences that may need other tests to evaluate the safety before being introduced in use.

Desirable characteristics of casein peptides with simultaneously enhanced emulsion forming ability and antioxidative capacity in O/W emulsion

This study aimed to explore the desirable characteristics of casein hydrolysates with simultaneously enhanced emulsion forming and antioxidative ability in oil-in-water (O/W) emulsion. Effects of physicochemical properties of casein hydrolysates prepared by trypsin, alcalase, and neutrase on physical and oxidative stability of O/W emulsion were studied, and potential antioxidant peptides were characterized by UPLC-MS/MS and bioinformatics analysis. Results showed that casein hydrolysates at a lower degree of hydrolysis (DH) produced by trypsin displayed higher zeta potential absolute values (−31.7 mV), smaller particle size (170.75 nm) and improved antioxidant properties compared to alcalase and neutrase, and could form more stable emulsion with d4,3 of 0.39–0.41 μm at a broader range of DH. Additionally, compared with sodium caseinate (NaCas), the oxidation of emulsion was significantly retarded during the storage progress prepared by the trypsin hydrolysates at DH of 4.49% alone or with Tween 20. Peptidomics indicated that the trypsin hydrolysates contained more peptides with basic amino acids at C-terminal and aromatic amino acids at N-terminus, which exhibited cationic amphiphilic properties with grand average of hydrophilicity values (GRAVY) of −0.5 ∼ +0.5 and pI > pH 7.0. This could contribute to their better emulsion formation and inhibition of lipid oxidation ability by producing dense interfacial layers with peptides interactions or excluding metal ions in O/W emulsions. These results suggested that casein hydrolysates at lower DH prepared by trypsin owned potential amphiphilic antioxidant peptides, which were desired to enhance the stability and functional activity of O/W emulsion.

The effect of casein hydrolysate intake on cerebral neural regulation during cognitive tasks in the elderly.

Human and animal studies have shown that casein peptide (casein hydrolysate) has positive effects on cognitive function. This double-blind randomized controlled study aimed to investigate whether single ingestion of casein peptide could affect cognitive function (executive function) and cognitive neural activity in healthy older adults. We assigned 47 participants to one of the three dietary supplements as follows: casein peptide (TMP, n=15), casein (TMC, n=16), and indigestible dextrin (TMF, n=16). Dietary supplements were ingested 30min before starting the experiment; moreover, neural activity while performing the task-switching reaction time (SWT) trial was assessed through functional magnetic resonance imaging (fMRI). Additionally, we used a visual analog scale (VAS) test to assess the pre- and post-test feeling and mood. Regarding the parameters of the SWT trial, there were no significant among-group differences in the reaction time, accuracy rate, and %SwitchCost. Contrastingly, the fMRI experiment revealed among-group differences in the main effects in the medial frontal gyrus, Supplementary motor cortex (SMC), posterior cingulate gyrus (PCg), and amygdala (Amyg). Specifically, there was a significant decrease in the neural activities in the SMC and PCg in the TMP group than in the other two groups. Moreover, there was a significant increase in the neural activity in the Amyg in the TMP group compared with the TMF, but not the TMC, group. Furthermore, the VAS score was significantly higher in the TMP group than in the other two groups. There were no recorded adverse outcomes. Our findings suggested that TMP ingestion by older adults could temporarily suppress complementary neural activity in specific brain regions involved in executive functions, as well as default mode network activity, which could improve cognitive neural activities.

Differentiating ultra-high temperature milk and reconstituted milk using an untargeted peptidomic approach with chemometrics

An untargeted peptide profiling based on ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry with chemometrics was performed to differentiate ultra-high temperature processed milk and reconstituted milk. Thirty-three marker peptides were identified, primarily released from the C- or N-terminal of β-casein and αs1-casein. These peptides were produced by heating and protease hydrolysis. Additional heating and storage time experiments showed that the level of 18 marker peptides increased with heat load and storage time, whereas 15 peptides were solely influenced by heat load. The peptides from β-casein showed higher sensitivity to thermal stress compared to those from αs1-casein. Additionally, eight modified peptides of casein were identified as indicators of milk thermal processing. The identified marker peptides can distinguish ultra-high temperature processed milk and reconstituted milk, and are suitable for monitoring heating processes and storage of milk.

Structural characterisation, gastrointestinal digestion stability and transepithelial transport study of casein peptide–zinc chelate

SummaryIn this study, a promising nanoparticle of casein peptide–zinc chelate (CNP‐Zn) for zinc supplementation was obtained. The structure properties and transepithelial transport mechanism were investigated. The amino acid composition, particle size, zeta potential and microstructure were significantly changed after chelation with zinc ions. The results of fourier‐transform infrared spectroscopy suggested that the oxygen atoms from both the carboxyl and carbonyl groups, amides, were responsible for zinc chelation. After gastrointestinal digestion, over 40% of zinc remained bound in CNP–Zn. The transepithelial transport results showed that paracellular route and PepT1 transporter were involved in the absorption of CNP–Zn, and almost 50% of the bound zinc was co‐transported with peptides. These findings would provide a relatively comprehensive cognition for developing efficient dietary zinc carriers.

Occluding dentin tubules with monetite paste in vitro.

This study was performed to evaluate the occlusion of monetite paste on dentine tubule and provide a new potential method for treating dentine hypersensitivity. Calcium oxide, strontium chloride, and polyethylene glycol phosphate were mixed in a certain proportion and ground in a planetary ball mill. The reaction was carried out by adjusting the pH to obtain monetite and hydroxyapatite paste. The morphological characteristics of the paste were observed through scanning electron microscope (SEM). The structure and composition were analyzed through X-ray diffraction (XRD) and Fourier transform infrared spectrometer (FTIR). The extracted third molar was selected to undergo demineralization to establish the in vitro study model of dentin hypersensitivity. The samples were randomly divided into four groups: blank control group (treated with distilled water), casein peptide phosphate-amorphic calcium phosphate (CPP-ACP) group, monetite paste group, and hydroxyapatite paste group. Each group was used to scrub the dentin surface with the corresponding materials for 7 days. The morphological characteristics of the dentin surface and section were observed through SEM, the microhardness of the dentin before and after mineralization was analyzed with a microhardness tester, and the composition of the deposits on the surface of the mineralized samples was examined through XRD. XRD and FTIR showed that the composition of the paste was mainly monetite, and the composition of hydroxyapatite paste was mainly composed of hydroxyapatite. SEM revealed that the size of the crystal particles of the synthesized paste was tens to hundreds of nanometers. Monetite and hydroxyapatite paste could produce a thicker mineralization layer on the dentin surface, and the mineralization of the dentin tubules of monetite was deeper than that of hydroxyapatite paste. The microhardness of the monetite paste group was significantly less than those of the hydroxyapatite paste groups (P<0.05). Monetite paste could effectively block the exposed dentin tubules and be used for treating dentin hypersensitivity.