• Food Science of Animal Resources
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• v.30 no.3
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• pp.443-448
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• 2010

Casein phosphopeptide (CPP) enhances calcium absorption in humans. Lactic acid bacteria (LAB) are capable of synthesis of cell-surface proteinase, which can hydrolyze milk protein and release several types of peptides in the medium. This study was conducted to characterize proteinase of LAB and to evaluate the CPP production from bovine milk. The content of CPP of milk produced by cell-free extract of LAB was determined based on the quantity of decomposed peptide from casein using the O-phthaldialdehyde (OPA) method. The proteolytic activity of LAB was assayed using fluorescein isothiocyanate (FITC)-labeled casein. Casein appeared to be a better substrate than whey proteins for extracellular proteinases of LAB. During fermentation, milk proteins were hydrolyzed by extracellular proteinase of LAB, resulting in an increase in the amount of free $NH_3$ groups. Overall, the results presented here indicate that CPP produced by LAB may be a promising material for novel applications in the dairy industry.

• Food Science of Animal Resources
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• v.22 no.1
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• pp.55-58
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• 2002

The economical producing method of casein phosphopeptide (CPP) and the physicochemical properties related to the solubilization of calcium were studied. Firstly, The compositions of the purified CPP-III were 13.1% of nitrogen, 2.3∼2.4% of phosphate and the ratio of N/P was 5.4∼5.6. In consideration of economic aspects, the preparation method of the CPP- I and II which were lower purity than the CPP-III was established. The physico-chemical property of the CPP was compared with the enzymically dephosphorylated CPP. CPP and polyglutamate effectively inhibited the formation of insoluble calcium phosphates at physiological pH.

• Microbiology and Biotechnology Letters
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• v.21 no.5
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• pp.468-472
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• 1993

For the production of Casein Phosphopeptide(CPP) inhibiting the insolubility of calcium, 10% sodium caseinate was treated with 1.5% of protease from Streptococcus sp.. Optimal conditions and productivity for the CPP production, and properties of the CPP were compared with tryptic hydrolysates of sodium caseinate. Optimum conditions of pH, temperature and reaction time were 8.0, 50C, 4 hrs, respectively. Under these conditions the productivity of CPP was 23% and Molecular weight of CPP was ranged from 3, 000 to 17, 000. The results also showed that the insolubility of calcium was completely inhibited by using 1.5 times of CPP for the amount of calcium.

• YAKHAK HOEJI
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• v.52 no.5
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• pp.407-411
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• 2008

Phosphorylation plays the most important role in cell signaling mechanism. Various methods to identify the phosphorylation sites of proteins using tandem mass spectrometry (MS/MS) have been reported recently. Furthermore, the enrichment strategy such as Titanium dioxide ($TiO_2$) method should be combined with MS/MS analysis to effectively identify phosphorylation sites. It is necessary to optimize phosphopeptide-enrichment strategy, $TiO_2$ method in this study, due to the low amount of phosphorylated form followed by analyzing them by MS/MS. To evaluate the several conditions to enrich phosphopeptides using $TiO_2$ method, we used ${\apha}$-casein as a standard phosphoprotein and analyzed a representative phosphopeptide (VPQLEIVPNpSAEER) peak of MS spectrum. Batch is better than column method for binding and 300 g/l DHB in loading buffer is better than lower concentration of DHB. 3% TFA and pH 10.5 shows high efficiency of phosphopeptide-enrichment for washing and elution steps, respectively. Finally we identified various efficient conditions of phosphopeptide-enrichment method using $TiO_2$. This optimized method would assist in reliable identifying thousands of phosphorylation sites existed in low abundance from various complex proteins.

• Microbiology and Biotechnology Letters
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• v.44 no.1
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• pp.68-73
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• 2016

Lactic acid bacteria showing both protease activity and the capacity to produce casein phosphopeptide (CPP) were isolated from Korean kimchi, a traditional food made from fermented vegetables. Among the 450 strains of isolated lactic acid bacteria, the strain MG-379 showed high protease activity and the highest ability to produce CPP. Characterization results showed that MG-379 was gram-positive and measured $0.6-0.8{\mu}m$ in diameter. DNA sequencing of MG-379 and comparison with other sequences using BLAST revealed a 100% identity with the sequence of Enterococcus faecalis. However, MG-379 showed a higher CPP-producing ability than E. faecalis KCCM 40450. Accordingly, MG-379 was newly named as E. faecalis MG-379. Amount of free calcium liberated by CPPs was 2227.5 and 1151.6 mg/kg for E. faecalis MG-379 and E. faecalis KCCM (control), respectively.

• Bulletin of the Korean Chemical Society
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• v.33 no.10
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• pp.3298-3302
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• 2012

Although offline enrichment of phosphorylated peptides is widely used, enrichment for phosphopeptides using $TiO_2$ is often performed manually, which is labor-intensive and can lead to irreproducible results. To address the problems associated with offline enrichment and to improve the effectiveness of phosphopeptide detection, we developed an automated online enrichment system for phosphopeptide analysis. A standard protein mixture comprising BSA, fetuin, crystalline, ${\alpha}$-casein and ${\beta}$-casein, and ovalbumin was assessed using our new system. Our multidimensional system has four main parts: a sample pump, a 20-mm $TiO_2$-based column, a weak anion-exchange, and a strong cation-exchange (2:1 WAX:SCX) separation column with LC/MS. Phosphorylated peptides were successfully detected using the $TiO_2$-based online system with little interference from nonphosphorylated peptides. Our results confirmed that our online enrichment system is a simple and efficient method for detecting phosphorylated peptides.

• Restorative Dentistry and Endodontics
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• v.44 no.3
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• pp.24.1-24.1
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• 2019

• Journal of Dairy Science and Biotechnology
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• v.36 no.3
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• pp.164-170
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• 2018

The objectives of this study were to manufacture casein phosphopeptide (CPP)/chitosan oligosaccharide (CSO) nanocomplexes and to investigate the impacts of manufacturing variables, such as CPP concentration and pH, on their morphological and physicochemical characteristics. Transmission electron microscopy (TEM) and particle size analysis were used to assess the morphological and physicochemical properties of the CPP/CSO nano-complexes, respectively. Based on the images obtained by TEM, the spherical shapes of the CPP/CSO nanocomplexes ranged from 50 to 150 nm. As the concentration of CPP was increased and the pH was decreased, the average particle size of the nanocomplexes significantly (p<0.05) increased. The CPP/CSO nanocomplexes had a highly uniform distribution with a polydispersity index value of less than 0.3. In addition, they had a negative surface charge with a zeta-potential value between -17 and -26 mV. The CPP/CSO nanocomplexes showed good stability during the freeze-drying process. In conclusion, CPP/CSO nanocomplexes were successfully manufactured, and the CPP concentration and pH were found to be key factors that affected their morphological and physicochemical properties.

• Journal of Animal Science and Technology
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• v.48 no.5
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• pp.671-682
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• 2006

This experiment was conducted to investigate the effects of dietary Ca levels and some feed additives such as isoflavone and casein phosphopeptide (CPP) on eggshell quality and hatching egg production in aged egg-type breeder hens. A total of three hundred and sixty, 56-week-old Hy-Line Brown breeder hens were divided into six groups and fed experimental diets of two levels of Ca (3.3% or 3.6%) either with addition of 0.2% isoflavone, 0.5% CPP or devoid of all for 5 weeks. There were no significant differences in laying performances and settable egg production among the groups. Significant increases (P<0.05) in eggshell strength were observed with increasing dietary Ca and addition of isoflavone, but not with addition of CPP. Fertility and hatchability were not influenced by dietary Ca and addition of isoflavone or CPP. The treatment had few significant effects on tibial proximal compositions and breaking strength. The concentrations of Ca, P, estrogen and calcitonin in serum were not affected by the dietary treatments. These results indicated that relatively high level of dietary Ca in combination with isoflavone had a beneficial effect on improving eggshell quality in aged egg-type breeder hens. But hatching egg production was not affected by dietary isoflavone or CPP.

• Molecules and Cells
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• v.23 no.3
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• pp.340-348
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• 2007

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi-phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.