Digestion Of Β-casein Research Articles

Profiling the differential phosphoproteome between breast milk and infant formula through a titanium (IV)-immobilized magnetic nanoplatform

Breast milk (BM) fulfills the nutritional needs of infants and sets the standard for infant formula (IF). However, profiling the differential phosphoproteome between BM and IF remains unclear. Herein, a titanium (IV) (Ti4+)-immobilized magnetic nanoplatform (Fe3O4@GO@PDA-Ti4+) was constructed by self-assembly polymerization of dopamine on magnetic graphene oxide, followed by immobilizing Ti4+ through chelation for phosphopeptide enrichment. Fe3O4@GO@PDA-Ti4+ possessed outstanding selectivity (1/1000, a molar ratio of β-casein digests to bovine serum albumin digests) and favorable sensitivity (2.5 fmol/μL), along with rapid magnetic separation. Excellent phosphopeptide capture efficiencies were obtained for BM and IF using Fe3O4@GO@PDA-Ti4+ as an adsorbent coupled with liquid chromatography-mass spectrometry/mass spectrometry. There were 191 and 239 phosphopeptides found in BM and IF, respectively, with 36 phosphoproteins identified in both. However, BM and IF shared only 17 phosphopeptides and 4 phosphoproteins. The variation in the phosphoproteome between BM and IF provides valuable insights into the optimization of IF humanization.

The Effect of A2 Milk on Gastrointestinal Symptoms in Comparison to A1/A2 Milk: A Single-center, Randomized, Double-blind, Cross-over Study.

β-Casein, a major protein in cow's milk, is divided into the A1 and A2 type variants. Digestion of A1 β-casein yields the peptide β-casomorphin-7 which could cause gastrointestinal (GI) discomfort but A2 milk containing only A2 β-casein might be more beneficial than A1/A2 (regular) milk. The aim of this study was to evaluate the differences in GI discomfort after ingestion of A2 milk and A1/A2 milk. A randomized, double-blind, cross-over human trial was performed with 40 subjects who experienced GI discomfort following milk consumption. For each intervention period, either A2 milk first (A2→A1/A2) or A1/A2 milk was first consumed for 2 weeks (A1/A2→A2) following a 2-week washout period. GI symptom rating scale (GSRS) scores, questionnaire for digestive symptoms, and laboratory tests including fecal calprotectin were evaluated. For symptom analysis, generalized estimating equations gamma model was used. A2 milk increased bloating (P = 0.041) and loose stools (P = 0.026) compared to A1/A2 milk in GSRS. However, A2 milk caused less abdominal pain (P = 0.050), fecal urgency (P < 0.001) and borborygmus (P = 0.007) compared to A1/A2 milk in questionnaire for digestive symptoms. In addition, fecal calprotectin also decreased or less increased after consumption of A2 milk compared to A1/A2 milk (P = 0.030), and this change was more pronounced in males (P = 0.005) than in females. There were no significant adverse reactions during the trial. A2 milk alleviated digestive discomfort in Koreans following A2 milk consumption (ClinicalTrials.gov NCT06252636 and CRIS KCT0009301).

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

A nitrogen-doped graphene tube composite based on immobilized metal affinity chromatography for the capture of phosphopeptides

A novel immobilized metal affinity chromatography (IMAC) functional composite, mNi@N-GrT@PDA@Ti4+, was fabricated based on ultrathin magnetic nitrogen-doped graphene tube (mNi@N-GrT) after chelated Ti4+ with polydopamine, following as a magnetic solid-phase extraction sorbent for rapidly selective enrichment and mass spectrometry identification of phosphorylated peptides. After optimized, the composite exhibited high specificity in the enrichment of phosphopeptides from the digest mixture of β-casein and bovine serum albumin (BSA). The robust method presented the low detection limits (1 fmol, 200 μL) and excellent selectivity (1:100) in the molar ration mixture of β-casein and BSA digests. Furthermore, the selective enrichment of phosphopeptides in the complex bio-samples, was successfully carried out. The results showed that 28 phosphopeptides were finally detected in mouse brain, and 2087 phosphorylated peptides were identified in the HeLa cells extracts with specific selectivity of 95.6%. The enrichment performance of mNi@N-GrT@PDA@Ti4+ was satisfactory, suggesting that the functional composite provided a potential application in the enrichment of trace phosphorylated peptides from the complex biological matrix.

Design of reversibly charge-changeable rhodamine B modified magnetic nanoparticles to enrich phosphopeptides

In the present study, by employing ethylenediaminetetraacetic acid (EDTA), tetraethylene pentaamine (TEPA), and rhodamine B (Rb), we designed and synthesized a magnetic adsorbent (Fe3O4@EDTA@TEPA@Rb) on the basis of reversible charge change of Rb and applied to capture phosphopeptides. Rb existing in open planarized zwitterion form when stimulated by acidic loading buffer adsorbs negative phosphopeptides via electrostatic interaction. Under the stimulation of alkalic eluent, ring-closed structure of Rb is formed to elute the enriched phosphopeptides. TEPA containing rich amino groups is used as a crosslinking agent, which is also protonated in acidic loading buffer to bond phosphopeptides. Then phosphopeptides are eluted when TEPA deprotonates in alkalic eluent. Coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) detection, phosphopeptide signals originated from 0.4 fmol/μL β-casein digests were successfully detected. In addition, Fe3O4@EDTA@TEPA@Rb can also efficiently enrich phosphopeptides from skimmed milk, human serum and saliva samples (26, 4, 39 phosphopeptides, respectively), opening a new gallery for phosphopeptides-related analysis. In general, the developed adsorbent has the great potential for further application in the near future.

Monodisperse Ti4+-immobilized macroporous adsorbent resins with polymer brush for improved multi-phosphopeptides enrichment in milk.

Enrichment of phosphopeptides before mass spectrometry (MS) analysis is essential due to the limitations of low abundance and poor ionization efficiency in complex biological samples. Immobilized metal affinity chromatography (IMAC), especially titanium ion (Ti4+)-IMAC, has become a popular strategy for enrichment of phosphopeptides due to high selectivity and sensitivity. Conventional Ti4+-immobilized macroporous adsorption resin (MAR) fabricated by monolayer modification can preferentially capture mono-phosphopeptide over multi-phosphopeptides, which takes on more functions in the regulation of cell behaviors of organism. In this paper, a kind of monodisperse MAR microsphere with functional polymer brush (Ti4+-Brush@MAR) was prepared and modified via surface-initiated atom transfer radical polymerization (SI-ATRP). Compared with common Ti4+-MAR without polymer brush, Ti4+-Brush@MAR exhibited high enrichment specificity not only for mono-phosphopeptides but also for multi-phosphopeptides in β-casein or milk digest samples. As a result, a total of 93 unique phosphopeptides mapped to 18 phosphoproteins were identified from 5 μL milk, and the limit of detection is 10 fmol. It is expected that Ti4+-Brush@MAR would be utilized to enrich both multi-phosphopeptides and mono-phosphopeptides in additionalbiological or food samples.

Worldwide Research Trends on Milk Containing Only A2 β-Casein: A Bibliometric Study.

Simple SummaryA1 β-casein has been correlated with adverse health outcomes, and, as a consequence, milk containing only A2 β-casein has emerged on the market. There has been a relevant increase in publications in this area since 2010. Food Science Technology and Agriculture were the main research areas of this topic. The term β-casomorphin was the most frequently used. The USA, New Zealand, and Australia were the most productive countries, though the most productive research institutions were, in absolute terms, from India, France, and Germany. The majority of the most cited studies that refer to A2 β-casein and health were reviews, and a few clinical trials have also been published.The protein fraction of β-casein may play a key role in the manifestation of a new intolerance: milk protein intolerance. The most common forms of β-casein among dairy cattle breeds are A1 and A2 β-casein. During gastrointestinal digestion of A1 β-casein, an opioid called peptide β-casomorphin-7 (BCM-7) is more frequently released, which can lead to adverse health outcomes. For that reason, novel products labelled as “A2 milk” or “A1-free dairy products” have appeared on the market. In this context, a bibliometric analysis on A2 β-casein research was carried out through the Web of Science (WoS) database. The main objective of this work was to provide an overview of the state of the art in the field of β-casein A2 by analyzing the number of publications per year, trends in thematic content, the most frequently used terms, and the most important institutions and countries in the field. This bibliometric study showed that a greater effort is needed to determine the possible implications of this novel product for human health and the market.

Selective transportation and energy homeostasis regulation of dietary advanced glycation end-products in human intestinal Caco-2 cells

Advanced glycation end-products (AGEs) are a chemically heterogeneous set of modifications widely found in processed foods. Due to uncertain bioavailability, dietary AGEs regulate energy homeostasis through mechanisms that largely remain unclear. In this study, selective transmembrane transport of AGEs with different modification types from glycated β-casein digest were identified and compared. The results showed that only a few types of free and peptide-bound AGEs can easily cross the Caco-2 monolayers and thus exert their effects. A combination of biochemical assays, mitochondrial analyses, and comparative experiments identified that the effect of AGEs on cellular energy homeostasis comes mainly from their free fractions. Mechanistically, free AGEs arrest the mitochondrial differentiation and mtDNA repair by intervening in the function of thymidine phosphorylase, and interfering with mitochondrial energy production by inhibiting the activation of AMPK-SIRT6 signaling pathway. These results demonstrate mechanisms by which processed foods cause mitochondrial dysfunction and lead to dysfunctional energy homeostasis.

Design and fabrication of reusable core-shell composite microspheres based on nanodiamond for selective enrichment of phosphopeptides.

A kind of core-shell composite microsphere (CM) with nano-on-micro structure was synthesized via grafting amine-modified nanodiamonds onto the surface of monodisperse nonporous polymeric microsphere. In this way, the agglomeration of nanodiamond particles in the solutionwas avoided. After modification with pyrogallol groups, CM could chelate titanium ions (Ti4+) and thus be utilized as immobilized metal affinity chromatography (IMAC) sorbent to enrich phosphopeptides from biological samples. The resulting Ti4+-CM exhibited high enrichment efficiency and specificity to phosphopeptides. A total of 106 of unique phosphopeptides mapped to 29 phosphoproteins were clearly identified from 5 µL ofa milk digest after enrichment. Owing to the strong chelation between Ti4+ and pyrogallol ligands, the Ti4+is not releasedfrom thesorbent after completion of theenrichment process. As a result, the Ti4+-CM sorbent could be reused, and no significant loss of enrichment efficiency occurred even on the fourth run employing a β-casein digest as the sample. The strategy adopted presents a new way to prepare ahigh-performance reusable IMAC sorbent.

Magnetic mesoporous silica nanoparticles modified by phosphonate functionalized ionic liquid for selective enrichment of phosphopeptides.

In this study, new magnetic nanoparticles (denotated as Fe3O4@mSiO2-PFIL-Ti4+) have been prepared by immobilizing titanium ions with phosphonate functionalized ionic liquid (PFIL) on the wall of core-shell structured mesoporous nanomaterials. The resulting nanoparticles possess large specific surface area, strong hydrophilicity and fast magnetic response. The composites can capture traces of phosphopeptides from the tryptic β-casein digest (0.08 fmol), a digest mixture of β-casein and BSA (1 : 10 000, molar ratio) as well as a blend of β-casein digest and a great quantity of phosphorylated protein (β-casein) and non-phosphorylated protein (BSA) (1 : 2000 : 2000, mass ratio), respectively, showing excellent sensitivity, selectivity and size exclusion ability. Additionally, Fe3O4@mSiO2-PFIL-Ti4+ shows excellent steadiness and can be reused at least 12 times. Moreover, this material was successfully applied to enrich endogenous phosphopeptides from complex bio-samples, including human saliva and serum.

Development of dual-ligand titanium (IV) hydrophilic network sorbent for highly selective enrichment of phosphopeptides

A hydrophilic metal-organic network based on Ti4+ and dual natural ligand, tannic acid (TA) and phytic acid (PA), has been developed to enrich phosphopeptides from complex bio-samples prior to liquid chromatography-mass spectrometric analysis. Due to the strong chelation ability of TA and PA, abundant Ti4+ can be immobilized in the material, forming hydrophilic network by one-step coordination-driven self-assembly approach. The sorbent, TA-Ti-PA@Fe3O4, exhibited satisfactory selectivity for the phosphopeptides in the tryptic digest of β-casein, and can eliminate the interference components in 1000-fold excess of bovine serum albumin. The adsorption capacity of the sorbents for phosphopeptides was up to 35.2 mg g−1 and the adsorbing equilibrium can be reached in 5 min. The adsorbing mechanism has been investigated and the results indicated that the Ti4+ in forms of [Ti(f-TA)(H2O)4]2+, [Ti(f-PA)(H2O)4]2+ and Ti(f-PA)2(H2O)2 may play an important role in the adsorption process. The sorbent of the TA-Ti-PA@Fe3O4 has been applied to enrichment of the phosphopeptides in tryptic digest of rat liver lysate, and 3408 phosphopeptides have been identified, while the numbers of the identified phosphopeptides were 2730 and 1217 when the sample was enriched by the commercial TiO2 and Fe3+-IMAC kit, respectively. This work provides a strategy to enrich phosphopeptides from complex samples and shows great potential application in phosphoproteome research.

A simple method to generate β-casomorphin-7 by in vitro digestion of casein from bovine milk

Gastrointestinal (GI) digestion of bovine milk promotes release of biologically active peptides, including β-casomorphins (BCMs). Previous studies demonstrate release of BCM-7 by digestion of A1 β-casein (β-CN), but the conditions required to increase the amount of BCM-7 released during digestion are unclear. In this study, a simple in vitro digestion method employing pepsin, pancreatin and leucine aminopeptidase (LAP) enzymes was used to digest CN isolated from regular and A2 milk. LC-MS analysis revealed that CN isolated from regular milk digested with pepsin-pancreatin released BCM-7 and Val-BCM-7 over time. Additionally, LAP, which cleaves the N-terminal valine, increased the yield of BCM-7. In contrast, significantly less BCM-7 and Val-BCM-7 were released by digestion of CN isolated from A2 milk. This simple method for digesting bovine casein can now be used to provide a platform for future work aimed at better understanding the reported gut health benefits of A2 compared to A1 milk.

Graphene oxide-metal oxide nanocomposites for on-target enrichment and analysis of phosphorylated biomolecules.

The surface of matrix-assisted laser desorption/ionization mass spectrometry target is modified for improved signal strength and detection of analytes. The developed method includes on-target enrichment and detection of phosphopeptides/phospholipids using graphene oxide-lanthanide metal oxides (samarium, gadolinium, dysprosium, and erbium) nanocomposites. Enriched phosphopeptides are detected using material enhanced laser desorption/ionization mass spectrometry and phospholipids by laser desorption/ionization-mass spectrometry. Nanocomposites are prepared using graphene oxide with respective metal salts at high pH. They are characterized for nano-morphology, chemistry, porosity, composition, crystallinity, and thermal stability. Phosphopeptides enrichment protocol is developed and optimized for tryptic β-casein digest and that of phospholipids by phosphatidylcholine standard. Statistical analyses of phosphopeptides and phospholipids from milk show overlapping results for gadolinium, dysprosium, and erbium oxide nanocomposites. GO-Gd2 O3 has better enrichment efficiency and application as LDI material. Selectivity for GO-Dy2 O3 is 1:2500, for GO-Sm2 O3 is 1:3500, and 1:4000 for GO-Gd2 O3 . GO-Er2 O3 has a sensitivity of 25 fmol, whereas the highest sensitivity is down to 0.5 fmol for GO-Gd2 O3 . On-target enrichment is batch to batch reproducible with a standard deviation of<1, reduced time of enrichment to 10min, and ease of operation compared to solid-phase batch extraction. The developed method enriches serum phosphopeptides characteristic of cancer-related phosphoproteins.

Enhanced specificity of bimetallic ions via mesoporous confinement for phosphopeptides in human saliva

Phosphopeptides were of great significance in disease diagnosis and monitoring its dynamic changes. In this article, we proposed a more efficient method to synthesize a kind of bimetallic mesoporous silica nanomaterials (Fe3O4@mSiO2-PO3-Ti4+/Zr4+) and applied it to the analysis of phosphopeptides in human saliva samples based on IMAC technology. The chelation group was introduced into mesopores at the same time as the formation of mesoporous silica which significantly reduced the synthesis procedure and improved the synthesis efficiency. The as-prepared materials showed great sensitivity, selectivity and size-exclusion performance for phosphopeptides in standard β-casein digests. More importantly, the materials identified 85 phosphopeptides in disease saliva samples which provided a candidate choice in future clinical examination.

Flexible and hierarchical metal-organic framework composite as solid-phase media for facile affinity-tip fabrication to selectively enrich glycopeptides and phosphopeptides

Micro-tip-based solid-phase microextraction is considered as one of the green and powerful analytical sample preparation techniques, but its efficiency is severely hampered by some basic issues such as tedious fabrication, instability of sorbent bed, and blocking of the tip, especially for biological samples due to low permeability. These issues are tackled by introducing a flexible and hierarchical substrate in the microtip, having good mechanical strength and specific functionality to capture the desired biomolecules. Considering the well-ordered and flexible structure of melamine foam, it was used as a substrate and for hydrophilic interaction chromatography (HILIC). Metal-organic framework, due to its excellent characteristics, was grafted on its surface anchored by self-assembling polydopamine. The resulting material was characterized and packed in the tip by just pressing the material in the conical structure of the tip. This affinity tip established good and tunable permeability and was used to selectively enrich glycopeptides as well as phosphopeptides. The affinity tip demonstrated excellent performance to enrich glycopeptides and phosphopeptides with a low limit of detection up to 0.5 fmol μL−1 from tryptic digests of horseradish peroxidase and β-Casein, respectively, and was stable up to 5 rounds of enrichment. Moreover, this affinity-tip also exhibited high selectivity up to up to 1:1000 (HRP digest to BSA digest) for glycopeptides and 1:200 (β-Casein digest to BSA digest) for phosphopeptides and demonstrated several other fascinating characteristics such as; excellent size exclusion effect for the omission of large-sized proteins, modest backpressure, reproducibility, reusability, smooth enrichment, and successfully applied to a human saliva sample.

Simultaneous analysis of cellular glycoproteome and phosphoproteome in cervical carcinoma by one-pot specific enrichment

Cervical carcinoma is a kind of common gynecological cancer, the physiopathology of which is consanguineously connected with protein glycosylation and phosphorylation. Here, the magnetic binary metal oxide composites with the functionalization of hydrophilic tripeptide is designed and synthesized for simultaneous enrichment of glycopeptide and phosphopeptide in HeLa cells. Multiple kinds of metal sites realize more comprehensive enrichment of phosphopeptides compared with single metal, and ultra-hydrophilic property of glutathione further endows the composites with excellent affinity towards glycopeptides. The composites exhibited low detection limit (0.5 fmol/μL) and high selectivity (digests of β-casein and BSA, 1:500, m/m) for phosphopeptides, also showed good enrichment efficiency for glycopeptide including detection limit (0.1 fmol/μL) and selectivity (digests of HRP and BSA, 1:50, m/m). Meanwhile, the composites still possess outstanding enrichment capability towards phosphopeptide and glycopeptide after stored for two months or six consecutive times reuse. Eventually, 1177 phosphopeptides and 438 glycopeptides are identified simultaneously from 100 μg HeLa cell digests. As anticipated, potential biomarkers, such as heat shock protein beta-1, DNA topoisomerase 2-alpha, proliferation marker protein Ki-67 and 60 kDa heat shock protein are detected, suggesting its promising application in discovery and screening of cervical carcinoma.

Comparative Effects of Milk Containing A1 versus A2 β-Casein on Health, Growth and β-Casomorphin-7 Level in Plasma of Neonatal Dairy Calves.

Simple SummaryBovine milk generally contains two types of the milk protein β-casein, A1 and A2. Enzymatic digestion of the A1 type yields the opioid peptide β-casomorphin-7, which is suggested to adversely affect human and animal health. This study aimed to compare the effects of milk containing either homozygote A1 or A2 β-casein on health and growth parameters in 47 dairy calves during the first three weeks of life. Additionally, we studied, for the first time, the levels of intact β-casomorphin-7 in plasma of calves fed milk of alternative β-casein genotypes. Milk feeding of “A2-milk” led to a lower milk intake and a looser fecal consistency (higher prevalence of diarrhea) compared to “A1-milk”. Nevertheless, weight gains and end weights of calves of both feeding groups were similar, which might be caused by the associated higher protein content of milk containing the A2 variant. Intact β-casomorphin-7 was detected in plasma after A1- and A2-milk consumption, but was almost 5 times higher for A1-calves. In summary, A2-milk minimized the cleavage of the opioid peptide β-casomorphin-7 and might have advantages in the development of pre-weaned dairy calves.Research has shown that digestion of A1 β-casein (β-CN) affects gastrointestinal motility and opioid activity through the release of the peptide β-casomorphin-7 (β-CM7). In the case of the A2 variant, the cleavage of β-CM7 does not occur or occurs at a very low rate. Therefore, the aim of the study was to compare the effects of milk containing either homozygote A1 or A2 β-CN on health and growth parameters of dairy calves. Forty-seven neonatal calves (24 females, 23 males) of the breeds German Holstein (GH, n = 9), German Simmental (GS, n = 33) and their crossing (GH × GS, n = 5) were used in a 21-day feeding study. Fecal score (FS), respiratory frequency (RF), and rectal body temperature (BT) were recorded daily, whereas body weight was measured at birth and at day 21 to estimate the average daily weight gain (ADG). Additionally, blood was collected from calves three times during the experimental period and, for the first time, the respective plasma samples were analyzed for intact β-CM7. Consumption of A2-milk led to a lower daily milk intake (dMI) (p < 0.05). Furthermore, fecal consistency was softer for calves fed A2-milk (p < 0.05). Although 44% of A2-calves had diarrhea or revealed a tendency towards it (FS ≥ 3), A1-calves had a prevalence of 21%. Calves with a FS of 4 were offered an electrolyte solution and received a dietary food supplement for the stabilization of the fluid and electrolyte balance. Nevertheless, similar ADG and end weights (EW) of calves fed A1- or A2-milk (p > 0.05) indicate that A2-milk may compensate higher diarrhea rates and lower dMI due to the associated higher protein content. This is the first report of intact β-CM7 in plasma of calves fed milk of either A1 or A2 β-CN. Evidence from this study suggests that due to the change in the amino-acid sequence, A2-milk might be able to prevent or, at least, to minimize the cleavage of β-CM7 in calves.

Hydrophilic polydopamine-derived mesoporous channels for loading Ti(IV) ions for salivary phosphoproteome research

Salivary phosphoproteome holds great promise in clinic diagnosis. For profiling of salivary phosphoproteome, it is essential to develop efficient enrichment methods prior to mass spectrum (MS). Among developed enrichment strategies, immobilized metal ions affinity chromatography (IMAC) has exhibited outstanding performance. In this work, we report a coherent approach where polydopamine (PDA) is first utilized to form mesoporous structure through soft templating method, then chelated with Ti4+ to construct hydrophilic polydopamine-derived magnetic mesoporous nanocomposite (denoted Fe3O4@mPDA@Ti4+). In virtue of the merits including ordered mesoporous channels, appropriate superparamagnetism, and abundant Ti4+, the enrichment strategy based on Fe3O4@mPDA@Ti4+ combined with MS is employed for accurate identification of phosphopeptides in β-casein digest and human saliva. As expected, Fe3O4@mPDA@Ti4+ revealed a great selectivity (1:200) and a low detection limit (0.1 fmol μL−1) toward phosphopeptides. More importantly, the further successful capture of phosphopeptides from human saliva indicated the prominent potential of this method for seeking phosphopeptide biomarkers in further analysis.

Determination of Total and A1-Type β-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides.

Gastrointestinal digestion of A1-type β-casein is conducive to β-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type β-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. New analytical methods were developed and validated for total and A1-type β-casein in milk and milk-derived ingredients. Data on total and A1-type β-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography-mass spectrometry. The measurement includes all protein sequences (intact, modified, and partial) which are potential sources of β-casomorphin-7. Total β-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type β-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type β-casein in the β-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type β-casein relative to total β-casein. The methods described enable the monitoring of the A1-type β-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. The mass fraction of A1-type β-casein in a commercial β-casein standard was determined to enable its use as a calibrant.

The interaction mechanism of β-casein with oligomeric proanthocyanidins and its effect on proanthocyanidin bioaccessibility

The interaction mechanism between β-casein and five types of oligomeric proanthocyanidins, including procyanidins B1, B2, B3, A2, and C1 was investigated using spectroscopy and molecular docking. The fluorescence spectroscopy results showed that B1, B2, B3, and A2 can quench the intrinsic fluorescence of β-casein by static quenching and C1 by both dynamic and static quenching. All five proanthocyanidins can form a binding site with β-casein, and the corresponding binding ability was as follows: procyanidins B1 (KA= 1317 ± 159 L⋅mol−1) > B2 (KA= 759 ± 179 L⋅mol−1) > B3 (KA= 761 ± 72 L⋅mol−1) > C1 (KA=138 ± 48 L⋅mol−1) > A2 (KA=99 ± 6.1 L⋅mol−1). According to thermodynamic analysis and molecular docking, the binding forces of proanthocyanidins with β-casein were mainly induced by hydrophobic interactions, van der Waals forces, and hydrogen bindings. Circular dichroism and Fourier-transform infrared spectroscopy supported the changes in the secondary structure of β-casein induced by proanthocyanidins. The interaction between β-casein and proanthocyanidins did not significantly reduce the digestion of β-casein but increased the recovery rate of procyanidin B1 by 8.9% during in vitro gastrointestinal digestion. This study laid the foundation for the application of β-casein-proanthocyanidins complex in the food industry.