Impact of sequential enzymatic hydrolysis on antioxidant activity and peptide profile of casein hydrolysate.

ABSTRACT: This paper shows the potential of dual enzyme approach on antioxidant activity of casein hydrolysates. Casein was hydrolysed using the proteolytic enzymes alcalase, flavourzyme in isolation and in sequential order. Casein hydrolysates were evaluated for the degree of hydrolysis, antioxidant activity, molecular weight distribution patterns and peptide sequence. Casein hydrolysate produced by the sequential hydrolysis of alcalase and flavourzyme showed higher degree of hydrolysis and antioxidant activity as compared to hydrolysate obtained by individual enzymes. In size exclusion chromatograph of casein hydrolysate S3, peptides with molecular weight of 0.57 kDa share 12% area in total area of chromatogram which was 10 times higher than that of hydrolysate S1 and nearly half of that of hydrolysate S2. On subjecting to HPLC-TOF-ESI separation potential antioxidant peptides were identified. The peptide sequence VLPVPQ along with potential fragments was identified in hydrolysate S1 and S2 and HPHPHLS along with its potential sequence was identified in hydrolysate S1, S2 and S3. Sequential hydrolysis of casein showed better antioxidant activity and peptide profile in less duration as compared to the casein hydrolysate obtained by individual enzyme.

Encapsulation of grape seed extract phenolics using whey protein concentrate, maltodextrin and gum arabica blends.

Grape seed extract (GSE) contain phenolic compounds that decrease the proclivity to various chronic diseases such as several types of cancer and cardiovascular diseases. The objective of the present study was to investigate the encapsulation of GSE polyphenols and their characterization. For this study, whey protein concentrate (WPC), maltodextrin (MD) and gum arabic (GA) were evaluated as encapsulating materials. For the preparation of stable microcapsules different WPC:MD/GA (5:0, 4:1, 3:2 and 0:5) ratios were assessed using ultrasonication for different time periods (20-40 min) followed by freeze drying. Encapsulation efficiency, antioxidant activity, particle size, surface morphology and release mechanism were determined. The GSE microcapsules coated with WPC:MD/GA ratio of 4:1 and 3:2 with core to coat ratio of 1:5 and prepared by sonication for 30 min were found to have highest encapsulation efficiency (87.90-91.13%) and the smallest particle size with maximum retention of antioxidant activity. Under optimized conditions, the low level release (43-49%) of phenolic compounds resulted under simulated gastric condition and significantly (p < 0.05) increased (88-92%) under simulated intestinal condition. Thus the results indicated blending of MD or GA with WPC improved the microencapsulation of GSE.

Evaluation of casein & whey protein hydrolysates as well as milk fermentates from Lactobacillus helveticus for expression of gut hormones.

BACKGROUND & OBJECTIVES: Milk proteins play a beneficial role in the regulation of food intake, postprandial glycaemia and enteroendocrine hormone secretions and thus are receiving considerable attention for the management of metabolic inflammatory disorders such as type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate the efficacy of peptide/s obtained from milk proteins (casein and whey) as well as from the milk fermented with Lactobacillus helveticus as secretagogues for gut hormones and to purify and characterize the active peptides. METHODS: Effect of hydrolysates of casein protein (CP) and whey protein (WP) and L. helveticus fermented milk on the expression of proglucagon, pro-gastric inhibitory peptide (GIP) and cholecystokinin (CCK) genes was monitored by real-time quantitative polymerase chain reaction. The active glucagon-like peptide-1 (GLP-1) secretion was also quantitatively measured using ELISA. RESULTS: Hydrolysates of CP and WP as well as fermentates of L. helveticus induced the proglucagon, pro-GIP and CCK expression and secretion of GLP-1 in STC-1 (pGIP/Neo) cells. However, intact casein exhibited maximum GLP-1 secretion and proglucagon expression. Two active peptides (F5 and F7) derived from CP1 and WP3 hydrolysates having the ability to upregulate the GLP-1 secretion by 1.6 and 1.8 folds were obtained, and the mass was found to be 786 and 824 Da, respectively, as determined by electrospray ionization-mass spectrometry. However, no single active peptide from L. helveticus fermented milk could be obtained. INTERPRETATION & CONCLUSIONS: Casein as well as fermentates obtained from L. helveticus fermented milk showed higher potential for GLP-1 induction. These can be explored as novel therapeutics to T2DM effectively after demonstrating their in vivo efficacy in appropriate animal models.

Encapsulation of antioxidant peptide enriched casein hydrolysate using maltodextrin-gum arabic blend.

Antioxidant peptide enriched casein hydrolysate (AO-CH) are receiving increasing attention due to their potential as functional ingredient. Encapsulation of AO-CH using maltodextrin-gum arabic (MD/GA) as wall material could represent an attractive approach to overcome the problems related to their direct application. Encapsulation parameter were optimized using different ratio of core to coat and proportion of coating material (10:0, 8:2, 6:4) under varying pH (2-8) for encapsulation efficiency (EE).The preparation P3 resulted in maximum EE (87%) using core to coat ratio 1:20, at pH 6.0 with 8:2 MD/GA ratio. The encapsulated preparation showed reduced bitterness (p < 0.05) compared to the casein hydrolysate together with maximum retention of antioxidant activity (93%). Further, the narrow range of particle size, indicates their better stability and represents a promising food additive for incorporation in food.

Iron (II)-chelating activity of buffalo αS-casein hydrolysed by corolase PP, alcalase and flavourzyme.

Iron is a vital substance for human health which participates in many biochemical reactions. It also act as initiator for many harmful oxidative process. Buffalo αS-casein enriched fraction (80 %) was hydrolysed independently by corolase PP (H1), alcalase (H2), flavourzyme (H3) and sequentially by alcalase-flavourzyme (H4). After ultrafiltration (10 and 3 kDa) hydrolysates were analysed for their iron chelation activity using ferrozine. For H1 group of hydrolysates highest iron (II)-chelation activity (265.58 µM Fe(2+/)mg protein) was found after 8 h of hydrolysis for H2 (267.56 µM Fe(2+/)mg protein) and H3 group of hydrolysates (380.68 µM Fe(2+/)mg protein) after 6 h of hydrolysis. Sequential hydrolysis was not effective for iron (II)-chelation activity. 3 kDa fractions show higher iron (II)-chelation activity than 10 kDa fraction. Flavourzyme was more effective for generation of iron (II)-chelating peptides from buffalo αS-casein.

Patient-Reported Financial Burden of Treatment for Colon or Rectal Cancer.

Importance: The longitudinal experience of patients is critical to the development of interventions to identify and reduce financial hardship. Objective: To evaluate financial hardship over 12 months in patients with newly diagnosed colorectal cancer (CRC) undergoing curative-intent therapy. Design, Setting, and Participants: This prospective, longitudinal cohort study was conducted between May 2018 and July 2020, with time points over 12 months. Participants included patients at National Cance Institute Community Oncology Research Program sites. Eligibility criteria included age at least 18 years, newly diagnosed stage I to III CRC, not started chemotherapy and/or radiation, treated with curative intent, and able to speak English. Data were analyzed from December 2022 through April 2023. Main Outcomes and Measures: The primary end point was financial hardship, measured using the Comprehensive Score for Financial Toxicity (COST), which assesses the psychological domain of financial hardship (range, 0-44; higher score indicates better financial well-being). Participants completed 30-minute surveys (online or paper) at baseline and 3, 6, and 12 months. Results: A total of 450 participants (mean [SD] age, 61.0 [12.0] years; 240 [53.3%] male) completed the baseline survey; 33 participants (7.3%) were Black and 379 participants (84.2%) were White, and 14 participants (3.1%) identified as Hispanic or Latino and 424 participants (94.2%) identified as neither Hispanic nor Latino. There were 192 participants (42.7%) with an annual household income of $60 000 or greater. There was an improvement in financial hardship from diagnosis to 12 months of 0.3 (95% CI, 0.2 to 0.3) points per month (P < .001). Patients with better quality of life and greater self-efficacy had less financial toxicity. Each 1-unit increase in Functional Assessment of Cancer Therapy-General (rapid version) score was associated with an increase of 0.7 (95% CI, 0.5 to 0.9) points in COST score (P < .001); each 1-unit increase in self-efficacy associated with an increase of 0.6 (95% CI, 0.2 to 1.0) points in COST score (P = .006). Patients who lived in areas with lower neighborhood socioeconomic status had greater financial toxicity. Neighborhood deprivation index was associated with a decrease of 0.3 (95% CI, -0.5 to -0.1) points in COST score (P = .009). Conclusions and Relevance: These findings suggest that interventions for financial toxicity in cancer care should focus on counseling to improve self-efficacy and mitigate financial worry and screening for these interventions should include patients at higher risk of financial burden.

Preparation and characterization of iron-chelating peptides from whey protein: An alternative approach for chemical iron fortification.

Iron fortification of staple food is a strategy utilized worldwide to address the concern of dietary iron deficiency. However, traditional salt-based fortification methods have limitations with gastrointestinal stability and bioavailability. Iron chelating peptides from easily available and scalable proteins such as whey protein have been proposed as promising candidates to circumvent the above mentioned limitations by enhancing iron absorption and bioavailability. In this study, we report methods to produce whey protein derived iron-chelating peptides and describe their physicochemical characteristics. Peptides derived from whey proteins prepared by ultrafiltration of whey followed by hydrolysation were iron chelated to produce peptide-iron complexes. These complexes had a size of 422.9 ± 3.41 nm, chelated iron content of 36.42 µg/ mg protein, and a low zeta potential (-10.80 mV) compared to whey peptides. Spectra analysis using ultraviolet-visible absorption and Fourier transform infrared spectroscopy showed structural transformation indicating iron chelation. Mass spectrometric analysis using LC-MS/MS confirmed the presence of both hydrophilic and hydrophobic peptides in the complexes with sizes ranging from 275 Da to 1916 Da. Furthermore, reduction in the antioxidant property of peptides following iron complexing indicates iron chelation. Our results suggest that whey protein derived peptide-iron complexes can be used as a potential alternative for chemical iron fortificants for food products and also as iron supplements.

Bio-accessible milk casein derived tripeptide (LLY) mediates overlapping anti- inflammatory and anti-oxidative effects under cellular (Caco-2) and in vivo milieu.

Inflammation and oxidative stress are closely linked patho-physiological processes which occur concurrently in many diseased conditions. Recently, interdependence between these two processes explains the antioxidant paradox associated with failure to select appropriate agents required for prevention of diseases known to be induced by oxidative stress. Present study established the overlapping anti-inflammatory and anti-oxidative potential along with bio-accessibility of milk casein derived tripeptide (LLY). Tripeptide exhibited anti-inflammatory response under ex vivo conditions by suppressing (P<.01) mice splenocytes proliferation and modulating their cytokines (IFN-γ, IL-10 and TGF-ß) with improved phagocytosis of peritoneal macrophages. Conversely, tripeptide displayed extraordinary radical scavenging ability and cellular anti-oxidative potential using chemical assays and H2O2 induced oxidative stress model on Caco-2 cells. Under cellular assessment, on one hand tripeptide inhibited (P<.01) intracellular ROS generation and reduced MDA and protein carbonyls but on the other also increased (P<.01) the activity of anti-oxidative enzyme, catalase without much effect on SOD and GPx. This anti-oxidative potential was further established by studying relative expression of genes (Nrf-2 and Keap1) and Nrf-2 nuclear translocation associated with anti-oxidative signaling in Caco-2 cells. Bio-accessibility of tripeptide and its intact transport across Caco-2 cell monolayer was also found to be 1.72±0.22% through PepT1 mediated transport mechanism. Besides, tripeptide displayed strong anti-oxidative and anti-inflammatory potential under in vivo conditions in mice against ethanol induced oxidative stress by elevating (P<.01) liver GSH content and by decreasing (P<.01) the activities of anti-oxidative enzymes, MDA along with reduced expression of CYP2E1, PPAR-α, TNF-α and COX-2 genes than ethanol control.

Formulation and characterization of nanoencapsulated curcumin using sodium caseinate and its incorporation in ice cream.

In the present investigation, the preparation and characterization of a curcumin nanoemulsion with milk protein (sodium caseinate) and its incorporation into ice cream were undertaken. Among the different combinations, the most stable formulation was observed using milk fat (8%), medium chain triglycerides (2%), curcumin (0.24%) and sodium caseinate (6%) with a mean particle size of 333.8 ± 7.18 nm, a zeta potential of -44.1 ± 0.72 mV and an encapsulation efficiency of 96.9 ± 0.28%. The effect of different processing conditions (heating, pH and ionic strength) on the particle size distribution and zeta potential of the nanoemulsion was evaluated. During heat treatment, the particle size of the nanoemulsion was increased from 333.8 ± 7.18 to 351.1 ± 4.04 nm. The nanoemulsion was destabilized at pH 4.6 and the particle size increased above and below pH 5.0. However, there was a slight increase in the particle size with a change in the ionic concentration. The release kinetics data suggested that in simulated gastro-intestinal digestion, the nanoemulsion was stable against pepsin digestion (a 5.25% release of curcumin), while pancreatic action led to a 16.12% release of curcumin from the nanoemulsion. Finally, our formulation was successfully incorporated into ice cream and the sensory attributes were evaluated. No significant difference was observed in the scores of the sensory attributes between the control and ice cream prepared with a curcumin nanoemulsion. Moreover, the encapsulation efficiency of the curcumin incorporated into the ice cream was 93.7%, which indicates that it can withstand the processing conditions. The findings suggest that ice cream is a suitable dairy product for the delivery of lipophilic bioactive components (curcumin) which can be used for therapeutic purposes.

Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (P<0.05) of bound iron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source.

Severe Ulcerative Esophagitis Induced by Crizotinib Therapy.

Crizotinib is an oral tyrosine-kinase inhibitor that inhibits anaplastic lymphoma kinase (ALK) in gene-rearranged non-small cell lung cancer (NSCLC). In 2011, the Food and Drug Administration approved crizotinib for treatment of locally advanced or metastatic ALK-positive NSCLC. The crizotinib adverse events profile included esophageal disorders in 11% of patients treated during trial phases I, II, and III, but none of them had severe events. We describe the development of severe ulcerative esophagitis secondary to crizotinib therapy and the re-introduction of therapy at a lower dose without recurrence of esophageal symptoms.