佐藤 薫

Lactoferrin (LF) was investigated as a natural solubilizer of iron. Under neutral conditions, 1 mole of LF Solubilized 200 moles of iron. In a single dose oral toxicity study, 2.5 g iron solubilized by lactoferrin (FeLF) per kg body weight had no side effects on the rat gastric mucosa, While ferrous sulphate and ferrous citrate (at the same level with respect to iron) caused some serious injuries. FeLF was orally administered to rats for 28 days to investigate its potential toxicity. Haemoglobin content and its regeneration efficiency in rats fed with FeLF were superior to those observed with ferrous sulphate, which is the most frequently used iron supplement. The availability of FeLF in a rat model of iron-deficiency anaemia was demonstrated. LF is useful as a natural iron solubilizer for food products and neutraceuticals from the viewpoints of safety, bioavailability, and productivity. (c) 2005 Elsevier Ltd. All rights reserved.

Phospholipid (PL) extracted from bovine milk was tested for its emulsifying properties in conjunction with the reconstitution of cream using butter oil. PL from bovine milk dispersed in the oil phase was found to stabilize the cream, whereas PL extracted from soybean oil was found to solidify the cream. Different PL species purified from bovine milk PL were tested for their emulsifying properties. PC from bovine milk dispersed in butter oil was shown to stabilize the cream, whereas PE and sphingomyelin had no such effects. PC from soybean oil also was found to have emulsifying abilities. It was suggested that PC stabilized the reconstituted cream, regardless of its origin.

We developed a basic protein purification method combining affinity chromatography with large-scale ion exchange chromatography by using of a heparinized chitosan bead, Sulfonated Chitopearl 3L. Because the molecular weights and isoelectric points of the basic proteins such as lactoferrin, lactoperoxidase, and secretory component are similar, we investigated conditions for sequential separation of the proteins by changing pH and ionic strength of the elution solvents. The optimal loading volume of rennet whey applied to the Sulfonated Chitopearl 3L was 500 ml/g resin for the maximum yield of lactoferrin, lactoperoxidase, and secretory component. Secretory component was successfully eluted at pH 6.0 to separate it from lactoferrin and lactoperoxidase. Lactoperoxidase was eluted with 10 mM citric buffer (pH 4.0) containing 0.5 M sodium chloride. Although lactoferrin was eluted at pH 4.0, desorption of lactoferrin with 1.0 M sodium chloride at pH 4.0 was incomplete because of the tight adsorption of lactoferrin onto Sulfonated Chitopearl 3L at this pH. Thus, lactoferrin was eluted with 10 mM sodium hydrogen carbonate (pH 7.6) containing sodium chloride from 0.7 to 1 M.

In this study, the stability of a model cream was investigated using palm oil as sole fat source. It was found that the addition of monomyristoylglycerol (MM), monopalmitoylglycerol (IMP), and monostearoylglycerol (MS) to palm oil promoted the solidification of the model cream while the addition of monolauroylglycerol (ML) and monobehenoylglycerol (MB) had no such effect. The solid fat content (SFC) values of pure palm oil cream and palm oil with the above-mentioned additives were measured after cooling the cream from 60 degreesC to 5 degreesC. The SFC values in the cream with added MM, MP and MS were found to be lower than those of pure palm oil and palm oil with ML and MB. Fractionation of palm oil with the help of acetone resulted in a palm oil fraction containing a decreased amount of tripalmitin and 1,3-dipalmitoyl-2-oleoyl-glycerol (POP). It was found that the model cream prepared with this fraction and the above-mentioned monoacylglycerols remained stable even in the presence of MM, MP, and MS. From the above results, it was suggested that the agglomeration of tripalmitin and POP around MM, IMP, and MS, which were preferentially adsorbed at the oil-water interface of oil droplets in the model cream, led to a destabilization of the oil-in-water emulsion and to the solidification of the model cream. At the same time, it was suggested that the combination of the fatty acid moiety of monoacylglycerols with tripalmitin and POP played an important role in the destabilization of the model cream.

We have previously demonstrated that lactoferrin was incorporated into a lymphocytes and that a trypsin treatment for a short period reduced the number of lactoferrin molecules incorporated into B lymphocytes, An N-terminal sequence analysis revealed that the mild trypsin treatment had cleaved the three N-terminal amino acids, Gly(1)-Arg(2)-Arg(3). Chemical conjugation of lost sequence analogue Gly-Arg-Arg-Gly with the mildly digested lactoferrin recovered the interaction with a lymphocytes, while conjugation of acctyl-Arg-Arg-Gly, a deamino analogue of Gly-Arg-Arg-Gly, did not recover the interaction, This shows that the N-terminal basic region containing N-terminal Cry played an important role in the interaction with B lymphocytes. Acylation of the amino groups of lactoferrin also significantly reduced the interaction with a lymphocytes, and an O-methylisourea treatment of the amino groups, which preserved the positive charge, hardly affected the interaction. These results suggest that both the N-terminal basic region and the basic characteristics of the whole molecule contributed to its interaction with B lymphocytes.

Changes in the tryptic whey protein isolate (WPI) hydrolysate heated at 90 degrees C for 10 min were examined by gel permeation chromatography, SDS-PAGE and electronmicroscopy. Turbidity of the heat-treated WPI hydrolysates increased in relation to the increase in degree of hydrolysis (DH). The heat-treated WPI hydrolysates with a DH between 3.08 and 6.00% were of a milk-white color without any precipitates. The patterns of the heat-treated WPI hydrolysates on SDS-PAGE and the elution profiles obtained by gel filtration demonstrated preferential aggregation of beta-lactoglobulin in the tryptic WPI hydrolysates during heat treatment. On transmission electronmicroscopy the beta-lactoglobulin aggregates were similar to casein micelles in size.

Hydrolysis of whey protein isolate (WPI) solution prior to heat treatment induced preferential aggregation of beta-lactoglobulin, accompanied by oxidation of sulfhydryl to disulfide groups or sulfhydryl-disulfide exchange reactions. The degree of aggregation depended on the degree of hydrolysis (DH) and protein concentration of the hydrolysate on heating. Although beta-lactoglobulin could not be completely separated from alpha-lactalbumin, the optimum conditions to remove beta-lactoglobulin from WPI solution were a protein concentration of 5 % and a DH between 3.0 and 5.4 % prior to heat treatment.

After heating 10 % (w/w) of whey protein isolate (WPI) solution to 90 degrees C for 10 min, the partially heat-denatured WPI solution was subjected to proteolytic digestion with trypsin, papain, pronase, pepsin and protease at neutral pHs. The results indicate that 3-dimensional gel networks were formed after proteolytic digestion with trypsin, papain, pronase and protease within 8, 2, 4 and 1.3 h, respectively, but not with pepsin. Gelation was considered a result of hydrolyzation of the soluble aggregates of WPI following an interaction between them. These 4 proteolytic digestion-induced gels were generated in a similar mechanism, because similar SDS-PAGE patterns were obtained. The gel strength of the proteolytic digestion-induced gels depended on the pretreatment of heating temperature and the degree of hydrolysis attained.

塩化ナトリウム添加によって得られる加熱処理WPI溶液のゲルについて,その形成過程における疎水性度およびSH量の変化について検討した.<BR>10%WPI溶液を70℃以上で加熱した場合,高分子化したホエータンパク質の可溶性凝集体が得られる.この過程においてホエータンパク質の疎水性度は上昇し,SH量は低下することがわかった.また,電気泳動分析から可溶性凝集体はSS結合を介していることがわかった.すなわち,加熱処理によりホエータンパク質問で疎水性相互作用およびSH/SS交換反応が生じ,可溶性凝集体が形成されるものと推察した<BR>.20℃において加熱処理WPI溶液に塩化ナトリウムを添加した場合,疎水性度の急激な上昇とわずかなSH量の低下をともないながらゲルが形成されることがわかった.このことからゲル形成には疎水性相互作用およびSH/SS交換反応の両方が関与しているが,初期段階で疎水性相互作用の方がより大きく関与していると考えた.

WPI溶液を予め加熱処理し,室温で塩化ナトリウムを添加することによりゲル化する現象について検討し,以下のことが明らかとなった.<BR>(1) 80℃ 30分加熱処理した10% (w/w) WPI溶液に塩化ナトリウムを0.3-1.2% (w/w)添加すると,20℃において溶液は粘度の上昇をともないながら徐々にゲル化した.塩化ナトリウムの濃度が高くなるにしたがい粘度上昇は速く進み,ゲル化時間は短くなる傾向を示した.さらに,WPI溶液の加熱処理温度,塩化ナトリウムの濃度が高くなるにしたがい,ゲル強度は高くなることがわかった.<BR>(2) 電子顕微鏡観察の結果,WPI溶液を加熱処理することによりやや太く短い線状の可溶性凝集体が形成されることを確認した.さらに塩化ナトリウムの添加により,可溶性凝集体同士の会合が始まり,高分子化してゲルに至ることがわかった.塩化ナトリウム添加により得られたゲルは,GDL添加による酸性ゲルとよく似た網目状構造を呈していることがわかった.また,ゲルの網目状構造を形成している凝集体の太さは,塩化ナトリウム添加により得られたゲルの方がGDL添加による酸性ゲルよりもややランダムな構造をとることがわかった.