Casein phosphopeptides modulate calcium uptake and apoptosis in Caco2 cells through their interaction with the TRPV6 calcium channel

Dietary calcium intake is associated with colon cancer incidence due to its ability to modulate proliferation and apoptosis, cellular function directly linked to normal or tumour cell phenotype. In milk calcium is associated with casein whose hydrolysis produces the casein phosphopeptides (CPPs). CPPs induce calcium uptake in differentiated HT-29 and Caco2 cells. The aim of the present study was to explore: (i) the interaction between CPPs and the TRPV6 calcium channel in HT-29 and Caco2 cells; (ii) CPP effects on Caco2 cell functions. By reducing the expression of TRPV6 through small interfering RNA (siRNA), a decrease in cell response to CPPs was monitored in Caco2 cells (about 56%) but not in HT-29 cells. CPPs increased apoptosis both in undifferentiated and in transfected Caco2 cells. Based on the reported involvement of TRPV6 in cancer development, the results presented for CPPs may be helpful for their consideration as functional food ingredients.     

Casein phosphopeptides released by simulated gastrointestinal digestion of infant formulas and their potential role in mineral binding

Adapted and follow-up milk-based infant formulas were subjected to gastrointestinal digestion simulating physiological conditions. The naturally occurring casein phosphopeptides (CPPs) generated were fractionated by anion exchange high-performance liquid chromatography and sequenced by tandem mass spectrometry. In both infant formula digests, a total of 19 CCPs from bovine casein were identified, of which 7 corresponded to α_s1-casein and 12 to α_s2-casein. Most CPPs had the cluster sequence SpSpSpEE, representing the binding sites for minerals. The distribution of calcium, iron and zinc content in CPP fractions eluted from the anion exchange column was also studied. The results obtained suggest that calcium could be preferably bound to CPPs with the cluster sequence SpSpSpEE, whereas iron and zinc could be bound to CPPs containing the phosphorylated cluster and phosphoserine residues.     

Does the addition of caseinophosphopeptides or milk improve zinc in vitro bioavailability in fruit beverages?

The influence of caseinophosphopeptides (CPPs) added to fruit beverage versus milk based fruit beverage upon zinc retention, transport, and uptake, as well as the influence of Fe supplementation, were studied using a combined simulated gastrointestinal digestion/Caco-2 cell system.Zinc retention, transport, and uptake of milk based fruit beverage was 4- to 5-fold greater than that of fruit beverages with or without CPPs – no statistically significant differences being observed in relation to the presence or absence of CPPs. Possibly, a slow release of CPPs throughout the digestive tract, as can be expected to take place during the digestion of casein, has a more beneficial effect than the addition of preformed CPPs upon Zn availability.A significantly negative Fe × Zn interaction in relation to Zn retention and uptake was observed in fruit beverages. This should be taken into account in the formulation of such beverages, where the supplementing of both of these mineral elements is common practice.     

鲫鱼皮胶原肽对小鼠的促钙吸收作用

目的：研究胶原肽（collagen peptides,CPs）对小鼠的促钙吸收作用。方法：采用小鼠低钙膳食模型法, 以昆明雄性小鼠为实验动物,设置正常组、低钙模型组、碳酸钙组、CPs低剂量（500 mg/kg mb＋CaCO3）、高剂量 （1 000 mg/kg mb＋CaCO3）组、酪蛋白磷酸肽对照组,连续灌胃6 周,考察小鼠血清钙、磷、碱性磷酸酶（alkaline phosphatase,ALP）活力、钙吸收率、各项骨指标及骨微观结构、骨小梁形态变化。结果：CPs低、高剂量组小 鼠的ALP活力显著低于低钙模型组（P＜0.05）,其骨长度、干质量指数、骨钙含量、骨矿物密度（bone mineral density,BMD）、骨矿物含量（bone mineral content,BMC）均显著高于低钙模型组（P＜0.05）,CPs低剂量 组可使喂食低钙饲料的小鼠的各项指标达到正常组小鼠的水平。CPs低剂量组小鼠的ALP活力显著低于碳酸钙组 （P＜0.05）,其骨钙含量、BMD、BMC均显著高于碳酸钙组（P＜0.05）,提示补充碳酸钙的同时补充CPs,其促钙吸 收效果更好。结论：CPs可提高小鼠钙吸收率,增加骨小梁数目及强度,促进骨骼生长,从而能有效促进钙的吸收。     

CPPs阻钙沉淀活性与酪蛋白水解度之间关系的研究

通过对在不同的酶与底物浓度比下所得CPPs的特性进行研究,从而找出了在不同水解度下所得CPPs的阻钙沉淀性能与酪蛋白水解度之间的关系。即当酶与底物的比不同时,所得CPPs的N/P不同;N/P不同,其CPPs阻钙沉淀活性也不同。当N/P高时,CPPs的阻钙沉淀活性较低;N/P低时,CPPs的阻钙沉淀活性高。     

Stability and Kinetic Properties of a Magnetic Immobilized alpha-Amylase K

The properties of α-amylase K immobilized on hydrous titanium(IV)oxide coated magnetic iron oxide are reported and compared with the previously reported properties of the soluble form of the enzyme. The optimum pH was increased on immobilization but the addition calcium chloride caused a decrease. Compared to the soluble form the immobilized enzyme is less stable at 60 °C in calcium enriched buffers but more stable in calcium free buffers. The temperature-activity profile has a plateau between 40 °C and 60 °C attributable to a comformational change above 60 °C to a more active form. The action pattern in the hydrolysis of soluble starch was found to be unaffected by immobilization. Parameters affecting the amount of bound activity were studied. The magnetic recovery of the immobilized enzyme was complete.     

Production of Caseinophosphopeptides from Na-Caseinates Prepared from the Milk of Several Species by a Proteinase of Lactobacillus helveticus PR4

The aim of this work was to produce caseinophosphopeptides (CPPs) from Na-caseinates prepared from the milk of six species (bovine, sheep, goat, pig, buffalo, human) hydrolyzed by a partially purified proteinase from Lactobacillus helveticus PR4 and to characterize the CPP preparations by RP-FPLC (reversed-phase fast-protein liquid chromatography) and for calcium solubilizing/binding activity. The yield of CPPs ranged from 0.60 to 1.86% of the original proteins. The calcium-solubilizing activity varied from 2.5 (human CPPs) to 11.4 (buffalo CPPs) mg Ca²⁺ mg^-1 CPP. Moreover, minor differences in calcium-binding ability between the CPP from different species were observed. This work showed that CPPs with different calcium-solubilizing activity can be produced from various Na-caseinates by a Lb. helveticus proteinase. Milks having casein-fractions with similar amino acid sequences (e.g., sheep and goat), showed CPPs with the same activity. The data generated in this study show the potential of different caseins to yield CPPs and may help in the selection of milk for the production of functional foods enriched in CPPs.     

Changes in Size and Composition of Protein Aggregates on Heating Reconstituted Concentrated Skim Milk at 120oC

Treatment at 120^oC caused initial transfer of protein from the serum and small micelle fraction to the medium-sized micelle fraction. More extensive heating caused transfer of protein from the medium fraction to the large-sized micelle fraction, and the serum fraction as 'soluble’casein. Protein composition of large-sized micelle fraction remained essentially constant, but for the other fractions it changed throughout the heating period. Experiments with heated milks that had been made free of colloidal calcium phosphate (CCP) showed that a proportion of the calcium phosphate in milk, whether indigenous or precipitated by heating, became less soluble. This‘insoluble’calcium phosphate was possibly involved in joining the smaller protein particles into larger-sized aggregates.     

Cytochemical assessment of phosphopeptides derived from casein as potential ingredients for functional food

Phosphopeptides derived from casein hydrolysis are suggested to have beneficial effects beyond their mere nutritive value by enhancing availability of dietary minerals, especially calcium, iron and zinc. Apart from a possible positive action the potential may exist for adverse effects that could impose restrictions to their widespread application in functional foods for human nutrition. In the present work, various case-inophosphopeptide (CPP) preparations were assessed using different human cell culture model systems in order to estimate their cytotoxic potential and their influence on epithelial properties and differentiation of human intestinal cells (Caco-2). The general cytotoxic potential of CPPs was tested using the AlphaTox NR assay. Structural and functional differentiation of intestinal Caco-2 cells was determined by measuring transepithelial electrical resistance and activity of brush border associated alkaline phosphatase over an extended cultivation period. No loss in cell viability with respect to membrane integrity could be detected, as uptake and retention of neutral red dye (AlphaTox assay) into HeLa cells was not affected by the presence of CPPs. Cytochemical assays conducted on epithelial cells (Caco-2) showed no disturbance of normal cell development and differentiation with respect to structural as well as functional differentiation markers. As all CPPs tested did not provoke any adverse effects in terms of cytotoxicity and showed no impairment of cell differentiation and monolayer integrity of human intestinal cells, it may be supposed that CPPs do not provoke a cytotoxic response in vivo in the cells assayed in the present study.     

Effects of varying extracellular amino acid concentrations on bidirectional amino acid transport and intracellular fluxes in mammary epithelial cells

Understanding the regulation of cellular AA uptake as protein supply changes is critical for predicting milk component yields because intracellular supplies partly regulate protein synthesis. Our objective was to evaluate cellular uptake and kinetic behavior of individual AA when cells are presented with varying extracellular AA supplies. Bovine primary mammary epithelial cells were grown to confluency and transferred to medium with an AA profile and concentration similar to that of plasma from dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mM, which represented 16, 100, 186, and 271% of typical plasma AA concentrations, respectively, in lactating dairy cows. Twenty-four plates of cells (89.4 × 19.2 mm) were assigned to each treatment. Cells were first subjected to treatment medium enriched with ¹⁵N-labeled AA for 24 h and then incubated with treatment medium enriched with ¹³C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular medium free AA were analyzed for concentrations and isotopic enrichment using gas chromatography–mass spectrometry. A dynamic, 12-pool model was fitted to the data for 14 AA to derive unidirectional uptake and efflux, protein turnover, transamination, oxidation, and synthesis. The derived concentration for half the maximal uptake (k_m) indicated no saturation of AA uptake at typical in vivo concentrations for 11 of the 14 AA. Arginine, Pro, and Val appeared to exhibit saturation kinetics. Net uptake of all essential AA except Phe was positive across treatments. Most nonessential AA exhibited negative net uptake values. Efflux of AA was quite high, with several AA exhibiting greater than 100% efflux of the respective influx. Intracellular pool turnover was rapid for most AA (e.g., 2 min for Arg), demonstrating plasticity in matching needs for protein translation to supplies. Intracellular AA concentrations increased linearly in response to treatment for most AA, whereas 9 AA exhibited quadratic responses. Amino acid uptake is responsive to varying extracellular supplies to maintain homeostasis. No saturation of uptake was evident for most AA, indicating that transporter capacity is likely not a limitation for most AA except possibly Arg, Val, and Pro in mammary epithelial cells.     

Commercial Mineral Enhanced Dairy By‐Products as Sodium Replacers,Antioxidants and Calcium Fortifiers in Sausages

Sausages are perceived as high in Na and with a too high Na:K ratio. Frankfurter type sausages are regarded as important contributors of sodium in the diet and thereby of health risks. Surplus products from the dairy industry are various mineral powders enriched in either potassium, calcium, or phosphate and include various amounts of lactose. Sausages were produced at 3 sodium levels (equivalent to 13, 15, and 17 g NaCl/kg sausage) using 4 different milk ingredients (a dried skimmed milk powder, a calcium enriched milk powder, a potassium enriched powder, and a lactose enriched powder). The sausages with added calcium and potassium enriched milk powders resulted in the hardest sausages when compared at the same sodium level. Milk mineral addition also produced whiter and less red sausages. No effect on rancidity after 6 wk at chill (4 °C) storage was observed by adding milk minerals, when compared with adding dried skimmed milk powder. A significant advantage of using these milk minerals in sausages is that the Na:K ratio can be reduced from an unhealthy (in this study 36) to a far healthier ratio ( ? 2) with limited or no taste changes. High additions of milk calcium (6 g/kg), where Ca‐phosphates prevail, added as milk mineral, had no influence on sensory bitterness or aftertaste as typically observed for CaCl₂ additions. Ca additions to sausages are presently presumed to be an advantage with respect to human nutrition.     

Solubilisation of calcium and magnesium from the marine red algae Lithothamnion calcareum

Marine minerals are a potential source of calcium and magnesium for nutritional supplementation. This study analysed the solubilisation of calcium and magnesium from the skeletal remains of Lithothamnion calcareum. Scanning electron microscopy analysis demonstrated a nonporous microstructure. Spectrophotometric determination showed that the calcium and magnesium contents were 30.01 and 6.22% (w/w), respectively. Solubilisation of calcium and magnesium was highly pH dependent. The temperature‐dependent solubilisation of calcium fitted the shrinking core model. The apparent activation energy for calcium solubilisation was 28.6 kJ mol^?1. Inclusion of caseinophosphopeptides (CPPs), casein‐derived mineral binding peptides, during the solubilisation of calcium and magnesium appeared to decrease the extent of calcium solubilisation at pH 6.0 and 8.0. The results herein have implications for the choice of optimal pH conditions for the sustained release of calcium and magnesium from marine mineral sources.     

Fatty acids do not stimulate enteroendocrine cells via particle sensing mechanisms

Lipid digestion and absorption are closely regulated to achieve maximal assimilation. Cholecystokinin (CCK) is the key regulator of the gastrointestinal responses to fat, and is secreted by enteroendocrine cells (EEC) in response to luminal free fatty acids. The mechanisms by which EEC sense fatty acids remain unresolved, but could be attractive targets in strategies aiming to alter food intake and digestion. Previous evidence suggested that aggregates of insoluble fatty acids were principally detected, rather than free fatty acids in solution. Supportive observations followed which demonstrated that latex microparticles of similar size to fatty acid aggregates also activated CCK secretion via a rise in intracellular calcium. However, in the current report we show that this effect of latex microparticles is not specific to EEC. Three other cell types exposed to the microparticles also responded with a rise in intracellular calcium, and this occurred independently of any ability to sense fatty acids. In addition, latex microparticles operate via entry of extracellular calcium whilst fatty acids stimulate release of calcium from intracellular stores. Therefore fatty acids do not act upon the same pathway as latex microparticles. Nonetheless activating nutrient sensing cells by non-nutrient luminal factors that are not absorbed from the gut remains a potentially novel means to manipulate gut function and satiety.     

Stimulation by Calcium of the Formation of Active Extracellular Proteinase by Pseudomonas fluorescens spp.

Pseudomonas fluorescens produced extracellular proteinase in mineral salts medium in response to the addition of small molecular weight compounds derived from skim milk. Further fractionation of these compounds on Sephadex G-10 gave a highly stimulatory fraction lacking in peptides or amino acids but high in calcium. Further studies showed that proteinase production in mineral salts medium containing calcium was 29 to 64% of that in skim milk. Zinc, manganese, and magnesium ions failed to substitute for calcium. At 20 and 5°C, 50 and 10 μM calcium, respectively, were required for half maximum synthesis. Several complex media supported variable growth and enzyme synthesis; however, this variability did not seem related to the calcium content of the medium. Calcium was required for the formation of active extracellular proteinase by a number of strains of Pseudomonas fluorescens when growing in mineral salts medium. These results suggest that milk proteins are not essential for proteinase synthesis by Pseudomonas fluorescens.     

A study of various chemical pretreatments to fractionate lipids from whey protein phospholipid concentrate

Dairy-derived lipids such as phospholipids (PL) have been gaining interest due to their functional and nutritional properties. Our research goal was to develop a separation process (nonsolvent based) to produce an enriched dairy lipid fraction from whey protein phospholipid concentrate (WPPC). Various chemical pretreatments (i.e., adjustment of pH, calcium, or temperature) were applied to rehydrated commercial WPPC solutions. These treatments were done on a bench-top scale to aid in the precipitation of proteins or PL. The chemically treated solutions were centrifuged and fractionated into the following 3 layers: (1) top fat layer, (2) supernatant in the middle zone, and (3) sediment at the bottom of the centrifuge tubes. The thickness and size of the layers varied with the treatment parameters. Compositional analysis of each layer showed that the proteins, fat, and PL always appeared to fractionate in similar proportions. The proteins in each layer were characterized using sodium dodecyl sulfate–PAGE under reducing and nonreducing conditions. Different proteins including whey proteins, caseins, and milk fat globule membrane proteins and lipoproteins were identified, and no specific type of protein had an affinity for either the top or bottom layer. All types of proteins were present in each of the layers after centrifugation, and there were no major differences in fractionation of the proteins between layers with respect to the chemical treatment applied. The microstructure of protein and fat in WPPC was investigated using confocal laser scanning microscopy. Dual staining of the rehydrated WPPC solution with Fast Green FCF (proteins) and Nile Red (lipids) showed the presence of very large protein aggregates that varied in size from 20 to 150 μm, with fat trapped within these aggregates. The confocal laser scanning microscopy images of liquid WPPC revealed fine strands of a weak protein network surrounding the fat globules. This indicated that there were specific interactions between the proteins, as well as between the fat and proteins in WPPC. Sodium dodecyl sulfate treatment was performed to understand the nature of the interactions between protein and fat. We found that about 35% of the fat present in WPPC was in the form of free fat, which was only physically entrapped within the protein aggregates. The remaining fat had some form of association with the proteins in WPPC. Other fractionation techniques would be needed to obtain an enriched dairy lipid fraction.     

The Impact of Malt Derived Proteins on Beer Foam Quality. Part I. The Effect of Germination and Kilning on the Level of Protein Z4, Protein Z7 and LTP1

The effect of barley germination and kilning on three putative beer foam-positive proteins was investigated by immunoblotting and ELISA procedures. These procedures involved the use of specific antibodies raised to purified lipid transfer protein 1 (LTP1)and the two protein Z forms, Z4 (BSZ4) and Z7 (BSZ7). The free fraction of BSZ4 and BSZ7, and all LTP1 were extracted by aqueous salt-solution from barley and malt. The addition of reducing agent allowed the extraction of bound BSZ4 and BSZ7. A previously undescribed fraction of BSZ4 and BSZ7, refered to as latent, was extracted with SDS and reducing agent. The barley combined fraction (free + bound fractions) was surveyed in 93 barley varieties to show that BSZ4 was the dominant isoform, on average constituting ?80 % of all protein Z. Considerable variation was observed between varieties in the level of LTP1 (502–1144 μg/g) and the combined fractions of BSZ4 (18–2136 μg/g) and BSZ7 (38–771 μg/g). The free fraction is expected to be more available for extraction into wort during mashing than the bound or latent fractions. The level of LTP1 did not change substantially during germination, but a significant proportion of the latent and/or bound protein Z fractions was converted into the free fraction. In the seven varieties studied the free fraction of BSZ4 and BSZ7 increased 149–300% and 49–141%, respectively. Proteolytic cleavage in the reactive site loop converts protein Z to heat- and protease-stable forms that survive the brewing process. During germination most of the free BSZ4 and 30–70% BSZ7 was converted to the cleaved form. Kilning was found to reduce the amount of protein Z and LTP1 that could be extracted by 10–30% and 7–37%, respectively, which is likely to be counter productive for foam quality. These results suggest that barley variety selection and optimisation of germination and kilning protocols during malting may be opportunities for improvement of beer foam quality.     

The effect of whey protein-enriched fractions on the physical and sensory properties of frankfurters

Four Beta-lactoglobulin (β-lg) enriched fractions containing different mineral contents were prepared and evaluated in frankfurters. Frankfurters were assessed for cook loss, water holding capacity (WHC), textural and sensory characteristics. The addition of the β-lg fractions reduced the cook loss (p<0.001) in comparison to the control (6.63% vs 3.98%). The fractions (β-lg 1 and 2) with the lowest calcium level significantly reduced WHC (p<0.01). The β-lg fractions had no detrimental effect on the sensory characteristics (p>0.05). All of the fractions increased the TPA value of hardness in comparison to the control (p<0.001) while the springiness decreased in the fractions (p<0.001) with the lowest mineral level. This study showed that the mineral composition of the β-lactoglobulin fractions affected cook loss, tenderness and hardness (TPA) of the frankfurters and the addition of the β-lactoglobulin enriched fraction did not affect the organoleptic quality of frankfurters in comparison to the control. This study shows the potential for next generation whey protein fractions and their application in meat products.     

生物活性肽─酪蛋白磷酸肽(CPPs)的研究、应用及展望

酪蛋白的基本功能是为生物体提供氨基酸和氨源。但是,人们似乎并没有注意到奶蛋白,尤其是酪蛋白,还是生物活性肽的主要来源。在离体和在体条件下,通过蛋白酶水解酪蛋白均得到一系列生物活性肽。这些活性肽按照其生物学作用有：促进营养吸收的,如：磷酸肽,类吗啡因子;饭后促激素分泌的,如：类吗啡因子;激活免疫活性的,如：免疫活性肽,酪激肽．类吗啡因子;以及神经性内分泌信号传递体,如：酪激肽。酪蛋白磷酸肽（CPPs）是可以促进钙质吸收的生物活性肽。已经发现CPPs不仅可以促进钙质吸收,还可以有效地促进锌、铁等的吸收,也可以防止蛀牙,可以用于牙膏、漱口液等。日本和德国已经把CPPs定为功能性食品。但是,到目前为止．中国在这方面只有很少的研究,所以该综述意在介绍CPPs研究的主要工作,并展望它的研究和应用前景。     

Caseinophosphopeptide enrichment and identification

Caseinophosphopeptides (CPPs) were generated via tryptic hydrolysis of sodium caseinate followed by heat treatment (75 °C) at pH 6.0, 7.0 and 8.0. Calcium aggregation, ethanol precipitation and gallium immobilised metal affinity chromatography (IMAC) were applied to enrich the CPPs. Nano‐liquid chromatography electrospray ionisation‐quadrupole time‐of‐flight tandem mass spectrometry was used for phosphopeptide separation and identification. The combination of gallium IMAC isolation with calcium and ethanol precipitation appears to be a useful tool in characterising the fate of CPPs in CN hydrolysates during thermal processing. CPPs appeared to be less susceptible to thermal modification when heat‐treated at pH 7.0 than at pH 6.0 and 8.0. Different extents of methionine oxidation occurred in the CPPs depending on heat‐treatment pH. The results herein may have important implications for the manufacture of CPPs and for the quality control of CPP products.     

The effect of metal ion content on the viscosity of gum ghatti

Cold water extraction of gum ghatti gives a soluble gum and an insoluble gel. The two fractions were shown by potentiometric titration to have the same acid equivalent weight and to be largely in the salt form, with less than 10% in the free acid form. Quantitative analysis of the two fractions for metal ion content showed that the gel fraction was largely a calcium salt. The soluble fraction contains calcium, potassium, sodium, and magnesium. Precipitation of the calcium ions reduces the viscosity but the original viscosity is not restored by addition of calcium ions.