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Background Bovine milk κ-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheesemaking and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP which is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. Objective The aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption. Methods Jejunal fluids were collected from three adult participants (two men and one woman, age: 27 ± 7, BMI: 23 ± 1) for three hours after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid phase extraction and identified by mass spectrometry-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. Results Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (less than 2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11-20 amino acids in length were predominant (accounting for 68-88% of the total peptide ion abundance) in jejunal fluids during 1-3 h post consumption. Conclusions This study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form-the CMP-derived fragments-rather than that of intact CMP.

The ability of bovine κ-casein-derived caseinomacropeptide (CMP) to exert bioactivity in the human gut depends on its digestive survival. Sampling from the human jejunum after feeding CMP and top-down glycopeptidomics analysis facilitates the determination of CMP survival. To reduce interference from non-target molecules in mass spectrometric analysis, CMP must be isolated from digestive fluid. To identify an optimal extraction method, this study compared the profiles of CMP extracted from feeding material (commercial CMP in water) and digestive fluid by ethanol precipitation, perchloric acid (PCA) precipitation, and ultrafiltration. Ethanol precipitation yielded the highest ion abundances for aglycosylated CMP and glycosylated CMP in both feeding material and jejunal samples. Notably, PCA precipitation yielded the highest abundance of partially digested CMP-derived fragments in jejunal samples. Overall, ethanol precipitation was the most effective among the methods tested for intact CMP extraction from jejunal fluids, whereas PCA precipitation was optimal for extraction of CMP fragments.

Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.

Pectin is a co-product from juicing and the physico-chemical quality is important to functionality of pectin in food and pharmaceutical applications. Juice and peel from Huanglongbing (HLB) control and infected Valencia oranges were analyzed. Infected fruit had smaller diameter, lower L* and b* values and highly variable a* values. Juice from infected fruit had higher pectinmethylesterase (PME) activity of 1.5 Units/mL, which retained higher activity during 15 days of storage at 4 °C, and juice had higher cloud loss. Also, the juice obtained from infected fruit had lower °Brix (8.5–8.9), higher titratable acidity (0.71–0.88%), and lower pH (3.45–3.75). Molecular weights of 154–157 kDa and degrees of esterification of 65–67% were similar. Pectin yield from dried peel was reduced by 6% in infected fruit to 4.18 g/100 g. Total neutral sugars in pectin from control fruit were higher, 84.1 μg/mg, compared to infected fruit, 60.7 μg/mg. Glucose, galactose, and arabinose, were higher in control fruit, 25.0, 40.8, and 9.7 μg/mg, compared to 14.5, 27.4, and 8.3 μg/mg in infected fruit. Viscosity was 0.015 Pa s, in 1% pectin from control or infected fruit (p > 0.05). Juice quality, including cloud stability, was negatively impacted by HLB, however pectin functionality was minimally impacted.

Pectins from highbush blueberry powder were sequentially solubilized in water, chelator, and sodium carbonate solvents and precipitated (ADF.WSF, ADF.CSF, and ADF.NSF) or not precipitated in ethanol (DF.WSF, DF.CSF, and DF.NSF) before dialysis and freeze-drying. Alcohol precipitation more effectively removed bound anthocyanins and low molar mass pectins from water- and chelator soluble fractions than dialysis alone. Blueberry water soluble fractions were high methoxyl pectins, rich in neutral sugars (36 to 38 mol%), and had high molar mass (7.01 × 105 to 2.71 × 106 g/mol). Blueberry chelator soluble fractions were low methoxyl pectins and rich in uronic acids (90 to 92 mol%) which were more linear and less branched structure than other fractions or commercial citrus pectin. The molar mass ranged from 1.59 × 106 to 2.06 × 106 g/mol. Water- and chelator soluble fractions exhibited pseudoplastic behavior (n

The effect of pectin composition on the binding selectivity to anthocyanins was investigated by Langmuir isotherms with two blueberry pectins, water soluble fraction (WSF) and chelator soluble fraction (CSF). Adsorption was four times higher between CSF and anthocyanins, than WSF and anthocyanins. Higher adsorption was attributed to the more linear, more negatively charged homogalacturonan region of CSF, compared to the more branched, neutral sugar rich WSF. The CSF binding sites ( q m a x ) to anthocyanins were estimated at 1413–1904 μg/mg pectin, which was about eight times higher than WSF binding sites. No difference was observed in binding affinity ( K L ) among blueberry pectins. WSF, rich in neutral sugar side chains, showed lower and more variable interaction with anthocyanins. Binding studies also showed that blueberry pectin-anthocyanins adsorption was highest at pH 3.0 and lowest at pH 4.0. Increasing concentration from 25 to 200 mM citrate-phosphate (CP) weakened the pectin-anthocyanins interaction between pH values 3.0 and 4.0. However, no significant difference in binding was observed at pH 2.6 with increasing CP. As pH values changed from 4.0 to more acidic pH, electrostatic interaction decreased while hydrophobic interactions increased between blueberry pectin and anthocyanins, and hydrogen bond likely remained constant. Putative health benefits of anthocyanins are probably influenced by small pH and ionic strength changes in food matrixes.