• KSBB Journal
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• 제23권1호
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• pp.59-64
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• 2008

Streptococcus faecalis var. liquefaciens를 calcium alginate gel에 고정화 후 CPP생산 가능성을 실험하였다. 균체를 회수하여 담체에 고정화하는 방법보다는 배양액 전체를 고정화하는 방법이 보다 높은 생산성으로 CPP를 생산하였다. Streptococcus faecalis var. liquefaciens 전세포 고정화 방법에 의한 sodium casenate로부터의 CPP 생산의 최적조건은 bioreactor부피대비 bead사용량이 30%, 반응온도는 $50^{\circ}C$, 반응 pH는 7.0, 기질의 농도는 10% 이었다. 또한, 고정화 균체를 이용한 연속적인 생산은 회분식 반응에서 설정된 최적 조건하에서 20% 수준의 CPP를 최소한 1개월 이상 연속적으로 생산할 수 있었다.

• Journal of Dairy Science and Biotechnology
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• 제40권4호
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• pp.173-181
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• 2022

Industrial-scale Ca-fortified Dangmyon was manufactured with added casein phosphopeptides (CPP) to increase Ca adsorption in the intestine. Very low P content in eggshell Ca (egg Ca) and Dangmyon could make it possible to indirectly measure CPP content in Dangmyon. Partitions of the whole P present in Dangmyon were made into sweet potato, egg Ca, and CPP. The CPP content was obtained by multiplying CPP per P by the amount of P partitioned into CPP. It was found that 88.46% of CPP was obtained. However, when milk Ca, which was much higher in P, was added to fortify Dangmyon with CPP, it was found that the CPP content was either under- or over-estimated. Care must be taken when a much higher content of P as a Ca source is selected using this method. It was discovered that the added Ca and CPP were retained after cooking at boiling temperature; therefore, Dangmyon could be an excellent Ca and CPP carrier for humans.

• Journal of Nutrition and Health
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• 제15권1호
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• pp.1-8
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• 1982

유당(乳糖)을 함유(含有)하지 않은 분유식(粉乳食)을 섭취한 흰쥐의 장관내(腸管內)에는 Casein함유식(含有食)을 섭취한 때와 마찬가지로 C.P.P.가 생성(生成)되며 이 C.P.P.에 의해 장관내(腸管內) 칼슘이 증가(增加)되며 소장하부(小腸下部)에서의 칼슘흡수(吸收)가 촉진(促進)되는 것을 밝혔다. 그러나 10% 또는 30% Lactose를 첨가한 경우, 위의 제(諸) 현상(現象)은 보이지 않았다. 이것은 장관내(腸管內)에서의 칼슘의 가용화(可溶化) 및 흡수(吸收)에 대 한 C.P.P의 촉진효과(促進效果)가 유당(乳糖)과는 독립적(獨立的)으로 일어나는 것임을 시사함이다.

• Molecules and Cells
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• 제23권3호
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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.

• Journal of Dairy Science and Biotechnology
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• 제41권3호
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• pp.149-156
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• 2023

Milk protein is often fractionated/concentrated by using various techniques in dairy industries. Among these techniques, ultrafiltration (UF) is particularly efficient at concentrating the casein fraction of milk protein. The objectives of this study were to produce casein hydrolysates by concentrating the casein fraction in skim milk using the UF technique and to investigate the chemical composition of the casein hydrolysates. The skim milk was concentrated using a UF laboratory test unit equipped with 10 kDa and 30 kDa membranes. After UF, the protein content of the milk was concentrated up to ~7.2% and the Ca was concentrated up to ~196 mg/100 g of milk. Trypsin was then added to the concentrated skim milk to produce the casein hydrolysates. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the casein fraction was not present after hydrolysis, indicating that casein in the milk had been hydrolyzed. The Ca content in the casein hydrolysates was much higher (p<0.05) compared to Ca content in commercial casein phosphopeptides (CPP) indicating that was acidified during the manufacture of commercial CPP. In conclusion, it seems that casein hydrolysates containing large concentrations of protein and Ca can also be made from concentrated UF milk without acidification or renneting.

• 한국미생물·생명공학회지
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• 제21권5호
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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.

• 약학회지
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• 제52권5호
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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.

• 한국미생물·생명공학회지
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• 제44권1호
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• pp.68-73
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• 2016

본 연구에서는 전국 가정집에서 제조한 김치로부터 분리한 유산균들 중에서 protease 활성 및 CPP 생산 능력이 가장 높은 균주인 strain 0301을 동정하였고 해당 균주의 특성과 CPP 생산 최적 조건을 조사하였다. 0301은 $0.6-0.8{\mu}m$ 크기의 gram 양성 구균이며, 16s rDNA 염기서열 분석 결과 Enterococcus faecalis MG-379로 동정 명명하였다. E. faecalis MG-379 균주를 2% fructose를 첨가하고 pH 6.0인 배지에서 37oC에서 36시간 배양하였을 때 CPP 생산 능력이 최대였으며, ICP를 이용하여 측정한 결과 2227.5 mg/kg으로, 비교 균주인 Enterococcus faecalis KCCM 40450에 비해 약 2배 가량 높은 CPP 생산 능력을 갖는 것으로 나타내었다.

• Bulletin of the Korean Chemical Society
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• 제33권10호
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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.

• Journal of Dairy Science and Biotechnology
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• 제38권3호
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• pp.161-167
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• 2020

Cheese pizzas fortified with casein phosphopeptide (CPP) and calcium were subjected to an in vitro digestion to assess whether CPP could prevent the precipitation of calcium. The total calcium content of the cheese pizzas was adjusted to 1,000 mg per pizza (~370 g) with the addition of calcium originating from eggshells. Two levels of trypsin-digested caseins (367 and 459 mg), with a CPP content of ~20%, were added to each pizza. The in vitro digested pizzas were then centrifuged and the supernatant was mixed with Na₂HPO₃ at 37℃ to estimate the possible soluble effect of CPP on calcium. After 24 h of reaction, the solution was centrifuged and the calcium content in the resultant supernatant was analyzed by inductively coupled plasma-atomic emission spectroscopy. One-way statistical analyses showed that CPP had a positive effect on the solubilization of calcium against phosphate (p<0.05). Cheese pizza supplemented with 459 mg of CPP powder was able to prevent precipitation of calcium by 98.8%, whereas no CPP-added cheese pizza solubilized 86.4% of the calcium. A sensory test was also carried out, revealing that panelists could not discern the bitter taste of the CPP added to the pizzas.