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The susceptibility of Listeria monocytogenes to inhibition and inactivation by lactoferricin, a newly isolated antimicrobial peptide derived from bovine lactoferrin present in cow's milk, was studied in laboratory media. Lactoferricin showed an effectiveness similar to that of many clinically useful antibiotics, causing complete inhibition of four strains of L. monocytogenes (serotypes 1b, 2, 3, and 4a) at low concentrations varying within the range of 0.3 to 9 μg/ml depending on the strain and the culture medium used. The effectiveness of lactoferricin against L. monocytogenes was not strongly affected by the presence of various carbohydrates or proteins but was somewhat diminished in the presence of various salts. The peptide showed potent activity over the pH range of 5.5 to 7.5. The effect of lactoferricin was lethal, causing a rapid loss of colony-forming ability with all four strains tested.

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.

A sensitive semi-micro column high-performance liquid chromatography with fluorescence detection method was developed for the determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MP) and amphetamine (AP) in human hair. 4-(4,5-Diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and 1-methyl-3-phenylpropylamine were used as labeling reagent and internal standard, respectively. These drugs were extracted from hair into 5% trifluoroacetic acid in methanol, and fluorescent labeled with DIB-Cl. The separation of DIB-derivatives was achieved on a reversed-phase semi-micro ODS column with an acetonitrile-methanol-water (30:40:30, v/v/v%) mixture as a mobile phase. The limits of detection at a signal-to-noise ratio of 3 for MDMA, MDA, MP and AP were 0.25, 0.15, 0.25 and 0.19 ng/mg, respectively. Precision of intra- and inter-day assay as the relative standard deviation were in the range 1.5-6.8% (n = 5) and 2.7-4.7% (n = 5), respectively. The proposed method was highly sensitive and able to detect MDMA and its related compounds in small amounts of hair sample, and could be applied to quantification of six abusers' hair samples.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.

Myeloperoxidase (MPO) derived from milk leukocytes and lactoperoxidase (LPO) secreted from the mammary gland have been identified previously in human colostrum. These peroxidases are known to play host defensive roles through antimicrobial activity. The goals of this study were to measure the peroxidase activity in mature human milk and to characterize the enzyme responsible for the activity. As determined using 3,3′,5,5′-tetramethylbenzidine as substrate, whey prepared from human milk samples obtained 1 and 5 months postpartum showed levels of peroxidase activity equivalent to 0.13 ± 0.18 and 0.24 ± 0.21 μg/mL bovine LPO (bLPO; n = 13), respectively. Whey from early milk was fractionated into two peaks of peroxidase activity by cation-exchange chromatography; the peroxidase in the first peak was sensitive to dapsone, which is an inhibitor of LPO, whereas the second peroxidase was not. Whey from mature milk showed only the first peak. Purified bLPO and MPO showed chromatographic behaviors that were similar to the first and second peaks, respectively. The dapsone-sensitive peroxidase from mature milk was further purified (952-fold from whey) by hydrophobic interaction chromatography. This preparation showed two bands with molecular masses of 80 and 90 kDa by polyacrylamide gel electrophoresis and immunoblotting using an antibody against bLPO. After deglycosylation, two distinct proteins with lower molecular weights were observed. Amino acid sequencing indicated that both of these proteins are LPO. These results provide evidence that LPO is present in mature human milk and that it is responsible for most of the peroxidase activity in mature milk.

牛乳の官能特性に及ぼす殺菌条件の影響を検討することを目的に各種殺菌牛乳については専門家パネルにより,また,市販牛乳については主婦パネルにより官能評価を行なった.結果は以下の通りである.(1) 殺菌による牛乳の物理化学的性状への影響はHTST=LTLT

In emulsions prepared using a membrane emulsification system, dispersion droplet diameter depends basically upon membrane pore diameter. For practical applications, it is necessary to select the appropriate type and concentration of emulsifiers, U and Je. For practical applications in the food industry, where large volume production is conducted, it is especially important to increase Je. When preparing an O/W emulsion, this can be carried out by adding emulsifier to the oil phase, and Je can be increased up to 10 times. In the preparation of a W/O emulsion, increasing Je can be accomplished using a hydrophilic membrane pre-treated by immersion in the oil phase. The dispersion droplet diameter is roughly controlled and Je can be increased about 100 times in comparison to using a hydrophobic membrane. This makes a membrane emulsification system practical for large-scale food application of a W/O emulsion. Without adding preservatives, a low fat spread with a fat content of 25 wt% was created using this system.

指標細菌の耐熱性パラメーターに基づき計算される殺菌効果と蒸気吹込み式殺菌機による実測値との間で差が生じる原因として,殺菌機の保持管における温度振動が,殺菌効果に与える影響について検討を行った.100l/hの実験機および2150l/hの実用機を用いて,保持管における温度振動が,殺菌効果に与える影響について検討を行い,次の知見を得た.1) 保持管内で蒸気の吹抜けを防ぐために,背圧弁によって制御し,保持管内圧を殺菌温度における蒸気圧より0.8kg/cmcm2以上高くする必要がある.2) 保持管温度の振動データーを基に殺菌効果を推算した結果,保持管温度の振動は殺菌効果を低下させる要因の一つであることが示唆された. 3) 商業的な目的で殺菌処理を行うためには,このタイプの殺菌機は,制御上の変動要因が多いため実際に耐熱性菌を液状食品に接種し,殺菌条件の妥当性を評価することが必要と考えられる.

膜乳化法による食品のO/Wエマルション調製において,膜細孔径の種類,油相の種類,乳化剤の種類,濃度,連続相循環流速,乳化圧力などの乳化条件を変えてエマルションを調製し,その結果から膜細孔径と分散粒子径の関係,単分散エマルションの調製に影響を及ぼす因子などについて検討した.また乳化速度を高める方法について検討した.(1) 油相(トウモロコシ油,ケロシン),乳化剤(ショ糖脂肪酸エステル,ポリグリセリン脂肪酸エステル,ドデシル硫酸ナトリウム)の種類によらず単分散性の高いエマルションが得られ,平均分散粒子径(Dp)は膜平均細孔径(Dm)に比例し,Dp=5.0Dmの関係にあった.またトウモロコシ油,SEを使用した食品系では,ケロシン,SDSを使用した場合と比較すると単分散性がやや劣る傾向にあった.(2) 単分散エマルションを得るには,乳化剤濃度,連続相循環流速,乳化圧力(乳化速度)が影響し,乳化剤濃度,連続相循環流速には連続相の乳化剤の種類より定まる下限値が存在し,乳化速度には上限値が存在した.(3) 単分散エマルションが得られる範囲でのDpの大きさは,ショ糖脂肪酸エステル濃度,連続相循環流速,乳化速度に依存しなかったが,乳化剤として蛋白質のカゼインNaを使用した場合,Dpの大きさはその濃度に依存した.(4) 乳化速度は乳化圧力の上昇にともなって増加するが,単分散エマルションを調製できる乳化速度の上限値は,水相,油相に添加する乳化剤の種類,濃度を適切に選択することによって高めることが可能であった.

アイスクリームの脂肪球凝集,官能検査によるドライネス評価およびメルトダウン試験に及ぼすフリージング時に使用するダッシャータイプとその回転数の影響について検討した.(1) ダッシャー容積率が高い程,およびその回転数が速い程1.2μm以下の脂肪球は減少し脂肪球の凝集は進み,7-15μmの巨大な脂肪球の凝集体の特異的な増加が見られた.(2) 15・Dよりもダッシャー容積率が高い80・Dの方が脂肪球の凝集は促進された.(3) 同一ダッシャーを使用するとき回転数が110rpmよりも220rpmの方が脂肪球の凝集が促進された.(4) アイスクリームの性状としてはダッシャー容積率が高い程,およびその回転数が速い程ドライネスが増加することが判明した.(5) アイスクリームの性状としてダッシャー容積率が高い程,およびその回転数が速い程メルトダウンのスピードは遅く保型性は優れていた.

A potent antimicrobial peptide, 'lactoferricin', was found to be generated upon gastric pepsin cleavage of lactoferrin. The active peptide consists mainly of a loop of 18 amino acid residues, derived from the N-terminal region of the lactoferrin molecule. Like various other antimicrobial peptides that display membrane-disruptive properties, it contains a high proportion of basic amino acid residues. A physiologically diverse range of micro-organisms was tested and found to be susceptible to inhibition by this natural peptide including Gram-negative and Gram-positive bacteria, yeasts and filamentous fungi. Its antimicrobial effect against sensitive micro-organisms was lethal. Electron microscopy studies revealed that it induces a profound change in cell ultrastructural features and causes substantial cell damage in bacteria and fungi. These findings suggest the possibility that active peptides of lactoferrin may have a role in the host defense against microbial disease. If produced in substantial quantities in vivo such peptides could have important physiological significance, especially in nursing infants.

A number of yeasts and filamentous fungi, including agents of skin disease (dermatophytes), were tested and found to be susceptible to inhibition by lactoferricin B. Effective concentrations varied within the range of 3 to 60 μg ml -1 , depending on the strain and culture medium used. Lactoferricin B inhibited fungal uptake of 1 H-glucose with effectiveness similar to polymyxin B, suggesting that it may target the cell membrane. It caused a profound change in ultrastructural features of the dermatophyte Trichophyton mentagrophytes

サワークリームの製造方法において,クリームの脂肪含有率,均質化圧力が物性に及ぼす影響をテクスチャー,離水,熱安定性などの観点から検討し,次の結果を得た.(1) クリームを均質化した時,脂肪球の平均粒子径は均質化圧力の対数に対して直線的に小さくなった.同じ均質化圧力では脂肪含有率が高いほど粒子径は大きかった.(2) サワークリームのテクスチャー解析から次のことが分かった.1) プランジャーの上下移動2mmでは非破壊の, 10mmでは破壊後のテクスチャーが測定できる.2) 硬度は非破壊,破壊の場合とも均質化圧力が高くなるほど上昇し,同じ圧力では脂肪含有率が高いほど硬度は高くなった.サワークリームをディップとして使う場合,ポテトチップなどですくって食べることが考えられる.この場合,ポテトチップが折れないことが条件となり,上下移動10mmでの硬度が2×104N/mm2以下が適していた.均質圧力の限界は脂肪含有率20%では約10-1MPa,30%では約5-1MPa, 40%では約1.5-1MPaであった.3) 凝集性については上下移動10mmの場合は,プランジャーにサワークリームが付着し1回目の上昇時にサンプルに穴があいてしまうため,測定値に信頼性が無かった.移動2mmの場合の凝集性は各脂肪含有率とも均質化圧力が高くなるほど減少し,同じ圧力では脂肪含有率が高いほど低い値を示した.また硬度が高くなるほど減少し,同じ硬度では脂肪含有率が高いほど凝集性は高くなった.4) 上下移動2mmの場合はサワークリーム表面の付着性が測定でき,均質化圧力を高くすると付着性は低下した.同じ硬度では脂肪含有率が高いほど高くなった.10mmでは,プランジャー側面,底面にサワークリームが付着し引上げられるためサワークリーム内部の付着性が測定でき,硬度が高いほど高い数値を示し,同じ硬度では脂肪含有率が高いほど高い数値を示した.(3) 離水の傾向は,同じ脂肪含有率では均質化圧力が高く硬度が高くなるほど減少した.同じ硬度では,脂肪含有率が高いほど減少した.ケーキのフィリングに用いる場合,ある程度の硬度と離水が生じにくいという条件が要求されるので,均質化圧力は高く,脂肪含有率が高いほうが望ましいことが分かった.(4) サワークリームを加熱した場合の熱安定性をカードの量から推定した.どの脂肪含有率とも1段目に10-1 MPa以上の圧力をかけるとカード量は急激に増え,料理のソース原料として加熱処理をするサワークリームでは, 10-1MPa以上の圧力をかけないほうが良いことが分かった.

圧力による芽胞の活性化を目的として, Bacillus属芽胞に加圧処理を施し,その活性化効果を検討した.得られた主要な結果は以下の通りである.(1) B. subtilis ATCC 6633を用いて牛乳中での加圧による活性化を検討したところ,加熱を用いた場合よりも良好な活性化効果が得られた.この効果は加圧温度の上昇及び加圧時間の延長とともに増大したが, 50MPaから200MPaまでの圧力変化に対しては顕著な差は見られなかった.(2) 加圧処理によって活性化されたB. subtilisの芽胞は大部分が保持中1時間以内に発芽し,耐熱性を失った.また,活性化されなかった芽胞は6時間の保持によっても発芽しない.非常に休眠性の高いものであった.(3) 加圧処理によって活性化されたB. subtilis芽胞の発育曲線は保持開始から緩やかな増加を示すが,対数増殖期の開始に要する時間については通常の発育曲線の場合と著しい違いはなかった.(4) 加熱による活性化が困難であったB. Polpmyxa NIAH-474及びB. licheniformis IAM-1l054の芽胞に対しても圧力による活性化効果が認められた.

アイスクリームの脂肪球凝集に及ぼすフリーザー出口温度とオーバーランの影響について検討した.(1) フリージングする前のアイスクリームミックス中の脂肪球は1.2μm以下が主であるが,フリージングすることにより脂肪球の凝集が進み,それらがフリーザー出口温度の低下と共に成長し保型性が良くなることが判明した.(2) オーバーランが高くなり気泡が多く取り込まれるに従って,脂肪球の凝集が進み,保型性も良くなることが判明した.(3) フリーザー出口温度の低下,およびオーバーランが高くなるにつれてアイスクリームのドライネスが増加した.さらに温度の低下,およびオーバーランが高くなる程この傾向が顕著になるのが認められた.(4) フリーザー出口温度の低下,およびオーバーランが高くなるにつれて脂肪球の凝集は進むが,脂肪球の凝集体の粒径も変化し,大きな粒径に移行した.脂肪球の粒度分布は1.2μm以下のものが減少し, 3～4μm, 8～15μmの脂肪球の特異的な増加が見られた.

The iron-binding properties of bovine lactoferrin in iron-rich solution were investigated. Ferrous iron was not stable in solution and was easily changed to the insoluble ferric state, but solubility of ferrous iron was stabilized by the presence of lactoferrin. However, casein hydrolysate or BSA was not effective in stabilizing iron in solution. As indicated by use of cibacron blue affinity gel, iron bound to lactoferrin, and the charge of supersaturated lactoferrin was higher than that of normal iron-saturated lactoferrin according to native PAGE electrophoresis. The evidence suggests that lactoferrin can bind iron at sites other than its chelate-binding sites, thereby stabilizing iron in solution.

パイプレスバッチプラント向けのレシピを用いた自律分散指向の運転管理システムの全体構造を提案した.個々の製品の生産手順をレシピとして記述し, それを単位作業に相当するサブレシピに分割・分散することで, 各ユニットが自律性を持った分散型の運転管理を効率良く行っていくために, 時間的パラメータの共通の取り扱い方の必要性を示し, 不確定幅を加味した所要時間の取り扱い方法を提案した.また, メッセージによる情報交換として, トラフィックマネージャを設置して, ユニットの移動や通路の利用を管理する方法と, 実際の生産の結果得られた具体的な時刻や実績値をインスタンスレシピ上に上書きして運転記録とし, 製品の動きと情報の動きを-体化する方法を示した.さらに, 納期を守ったり, プラントの稼働率を向上させるために, リアクターにより自律的に効率化する方法を探った.

パイプレスバッチプラント向けのレシピを用いた自律分散指向の運転管理システムを提案した.化学プラントの特性を反映した作業手順をレシピとして記述し, それを単位作業に相当するサブレシピに分割・分散することにより, ハードウェア (ユニット) とソフトウェア (サブレシピ) の一体化を計り, 各ユニットが自律性を持った分散型の運転管理を可能にした, さらに時間的・空間的に局所のユニット間での調整および同期によりプラント全体を動作させる方法を示した.また.レシピの複製を生成することによりレシピの汎用性を増すとともに知識と意思決定の分散化を可能にした.この結果.予期せぬ動作を起こさず安全であるという集中型の長所と, 保守が容易で負荷が1箇所に集中しにくい分散型の利点とを同時に実現することができた.

A cell-surface bound aminopeptidase from Lactobacillus helveticus LHE-511 was purified and characterized. The enzyme was found to have a monomeric structure and a molecular mass of 92 kDa. The optimal pH and temperature for activity were 7.0 and 37°C respectively. The enzyme was strongly activated by Co 2+ , completely inhibited by chelating reagents such as EDTA and 1,10-phenanthroline, and weakly inhibited by p -chloromercuribenzoate, suggesting that it is a metalloenzyme possessing a thiol group at its activity site. The enzyme showed high activity with p -nitroaniline derivatives or dipeptides and tripeptides that have a hydrophobic amino acid (Leu, AIa, or Phe) or diaminomonocarboxylic acid (Lys or Arg) at the N terminus.

An X-prolyl dipeptidyl aminopeptidase that hydrolyzed L-glycyl-L-prolyl- p -nitroanilide was purified from cells of Lactobacillus delbrueckii ssp. bulgaricus LBU-147 and characterized. The purified enzyme was homogeneous in electrophoresis analysis and appeared to be a trimer because the molecular weight was 270,000 by gel filtration and 90,000 by SDS-PAGE. The optimal pH and temperature for activity were 6.5 and 50°C, respectively. The enzyme was strongly inhibited by metal ions and chemical reagents such as Hg 2+ , Cu 2+ , Fe 3+ Fe 2+ , diisopropyl fluorophosphate, and p -chloromercuribenzoic acid, but it was weakly activated by sulfhydryl-group protective reagents such as 2-mercaptoethanol, dithiothreitol, and cysteine. These results indicated that this enzyme is a serine protease having its active site near a sulfhydryl group.

In order to develop a practical method for pasteurization of lactoferrin, the influence of pH on the heat stability of bovine lactoferrin was studied. Solutions (5%; wt/vol) of iron-free lactoferrin (apo-lactoferrin) in distilled water were adjusted to various pH values from 2.0 to 11.O and heated at various temperatures (80 to 120°C) for 5min. The heated samples were analyzed by HPLC and assayed for iron-binding capacity, antigenic activity, and bacteriostatic effect. The heated solutions of apo-lactoferrin gelled at neutral and alkaline pH. At acidic pH, lactoferrin in the heated samples remained soluble, and solutions were clear. The HPLC pattern of apo-lactoferrin heat-treated at pH 4.0 and 100°C was similar to that of unheated apo-lactoferrin. It resisted heating at pH 4.0 and 90°C for 5min without any significant losses of iron-binding capacity, antigenic activity, or antibacterial activity. These results indicate that apo-lactoferrin is very stable at pH 4.0 and high temperatures. It was considered that heating conditions of pH 4.0 and temperatures of 90 to 100°C for 5min were suitable as a practical method for pasteurization of lactoferrin. Moreover, the possibility of using an UHT method for more effective and efficient pasteurization of lactoferrin was demonstrated. Apo-lactoferrin treated at pH 2.0 or 3.0 and 100 or 120°C for 5min was apparently degraded, but the antibacterial activity was equal to or stronger than that of unheated apo-lactoferrin. This result suggests that some of the lactoferrin fragments produced by heat under acidic conditions have antibacterial activity.

To study the behavior of Bifidobacterium toward oxygen, oxygen uptake was investigated in detail. The cells of Bifidobacterial strains absorbed considerable amounts of oxygen. The exogenous oxygen uptake activity changed depending upon the period of incubation. Bifidobacterial cells also had high endogenous oxygen uptake, which was, in B. longum strains, as high as about 80% of the exogenous oxygen uptake activity. Bifidobacterial cells accumulated considerable amounts of polysaccharide, which was associated with cellular growth. By incubating the cultivated cells in a glucose-free medium, the endogenous oxygen uptake activity was decreased with a decrease of intracellular polysaccharide. Therefore it was postulated that the high endogenous oxygen uptake activity of Bifidobacterium was owing to the metabolism of intracellular polysaccharide. The enzymatic activity, which was involved in the mechanism of oxygen uptake, was also investigated.

A case of acute aortic dissection (AAD) presenting as sudden, transient paraplegia and severe back pain is reported. The patient was a 66-year-old male with a 10-year-history of hypertension. The pain characteristically migrated from the back to the neck and then returned to the back. He showed complete transverse myelopathy at the level of the 9th thoracic cord. Computed tomography disclosed internal displacement of aortic intimal calcifications, without abnormalities in the spinal canal, and myelography showed no spinal canal block or stenosis. Electrocardiography and chest x-ray indicated nonspecific changes of high amplitudes and mild cardiomegaly, respectively. Together, these findings suggested acute aortic dissection with spinal cord ischemia. The initial systolic blood pressure of 220 mmHg was lowered with medication, and the pain was controlled with morphine. He recovered fully and was discharged 80 days after the onset of symptoms, with no neurological deficits. AAD carries a very poor prognosis unless treated immediately. Therefore, it is very important to promptly differentiate this disorder from spinal vascular conditions that also produce back pain and paraparesis.

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 µL) were extracted by liquid–liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mm citric acid–20 mm Na2HPO4 aqueous buffer (pH 4.0)–CH3CN–CH3OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36–0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 ± 23 and 39 ± 6 ng/mL (Cmax), 20 ± 5 and 100 ± 10 min (Tmax), respectively. Copyright © 2006 John Wiley & Sons, Ltd.

Lactoferricin (LFcin) was initially identified as an antimicrobial peptide derived by pepsin digestion of lactoferrin (LF), a multifunctional innate-defense protein in milk. Various synthetic analogs of LFcin have also been reported. LFcin inhibits a diverse range of microorganisms such as gram-negative bacteria, gram-positive bacteria, yeast, filamentous fungi, and parasitic protozoa, including some antibiotic-resistant pathogens. LFcin kills target organisms by membrane perturbation and acts synergistically with some antimicrobial agents. LFcin exhibits numerous biological activities in common with those of LF. Whereas LFcin suppresses the activation of innate immunity by microbial components such as lipopolysaccharide (LPS) and CpG DNA, the peptide itself activates immunity. Administration of LFcin analogs has been shown to protect the host via direct antimicrobial activity and immunostimulatory effects in several infection models of mice. Here we present a comprehensive review of investigations of LFcin and related peptides.

Bovine lactoferrin is produced on an industrial scale from cheese whey or skim milk. The safety of purified lactoferrin has been confirmed from the results of a reverse mutation test using bacteria, a 13-week oral repeated-dose toxicity study in rats, and clinical studies. In order to apply active lactoferrin to various products, a process for its pasteurization was developed. Subsequently, lactoferrin has been used in a wide variety of products since it was first added to infant formula in 1986. A pepsin hydrolysate of lactoferrin is also used in infant formula. This hydrolysate contains a potent antimicrobial peptide named lactoferricin that is derived from the lactoferrin molecule by pepsin digestion. Semilarge-scale purification of lactoferricin can be performed by hydrophobic interaction chromatography. Lactoferricin also exhibits several biological actions and appears to be the functional domain of lactoferrin. Recent studies have demonstrated that oral administration of lactoferrin or lactoferricin exerts a host-protective effect in various animals and in humans. The results of these studies strongly suggest that the effects of oral lactoferrin are mediated by modulation of the immune system. Further elucidation of the clinical efficacy and mechanism of action of lactoferrin will increase the value of lactoferrin-containing products.Key words: bovine, lactoferrin, lactoferricin.

The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9+/-2.7 microg/ml (5.4+/-0.8 x 10(-6) mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7+/-0.7 x 10(-5) mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in the case of adults, from lactoferrin secreted into saliva.

We investigated the effects of lactoferrin (Lf)-related compounds on growth inhibition of Candida albicans by neutrophils or antifungal agents in vitro. Human neutrophils partially inhibited the growth of C. albicans. The growth inhibition caused by human neutrophils was augmented by the addition of human Lf at concentrations which did not show any inhibitory effect in the absence of neutrophils. Similar observations were obtained also with the following combinations: human neutrophils + bovine Lf, murine neutrophils + bovine Lf, and murine neutrophils + iron saturated bovine Lf, but not in the case of murine neutrophils + human transferrin. The minimum inhibitory concentration (MIC) of azole antifungal agents, clotrimazole, ketoconazole, fluconazole, and itraconazole was reduced by 1/4 to 1/16 in the presence of a sub-MIC level of each of bovine Lf, bovine Lf pepsin hydrolysate, and the antimicrobial peptide “lactoferricin® B” (Lfcin B). Other types of antifungal agents, amphotericin B, nystatin, and flucytosine did not show such combined effects with these Lf-related compounds. The anti-Candida activity of bovine Lf or Lfcin B in combination with clotrimazole was shown to be synergistic by checkerboard analysis. Clinically isolated azole-resistant C. albicans strains were more susceptible to bovine Lf or Lfcin B than azole-susceptible strains. Trailing growth of an azole-resistant strain in the presence of fluconazole was reduced by the addition of sub-MIC levels of bovine Lf or Lfcin B. These results suggest that Lf-related compounds even at relatively low concentrations may function as an antifungal effector in combination with neutrophils thereby modulating azole antifungal efficacies in vivo.

Recently we have reported that orally administered bovine Lf(bLf) exerts bacteriostatic effects against bacterial overgrowth in the intestine of specific-pathogen-free (SPF) mice fed milk. In this animal model, the in vivo bacteriostatic effect of bLf against the proliferation of intestinal Enterobacteriaceae, the bacteria most sensitive to bLf, was independent of the iron-chelating ability of bLf. In addition various proteolytic hydrolysates of bLf (with differing antibacterial activities in vitro) showed the same bacteriostatic effect as undigested bLf. These results suggest that the mechanism of in vivo bacteriostasis of Lf differs from the in vitro mechanism reported.

Lactoferricin (Lfcin®) is a bioactive fragment of lactoferrin derived from the bactericidal and putative lymphocyte receptor binding domain(s) located within the N-lobe of lactoferrin. Although known to be liberated from at least three species of lactoferrin, conditions leading to Lfcin generation in vivo and factors affecting its distribution are still not known. Recently, we have developed a method of surface-enhanced laser desorption/ionization (SELDI®) affinity mass spectrometry using n-butyl terminal groups for surface-enhanced affinity capture (SEAC) to quantify not only Lfcin generated in vivo but also other lactoferrin fragments. Unlike previous efforts to detect lactoferrin and Lfcin with specific antibodies, the SELDI affinity assay distinguished lactoferrin, lactoferrin fragments, Lfcin and unrelated peptides without their interference with each other. To evaluate Lfcin generation in vivo, the experimental design involved feeding 200 mL of 10 mg/mL (1.22 × 10−4 mol/L) bovine lactoferrin to an adult. Gastric contents were recovered 10 min after ingestion. Lfcin produced in vivo was directly captured by the SEAC device. The amount of Lfcin in the gastric contents was 16.91 ± 2.65 sg/mL (5.350 ± 0.838 × 10−6 mol/L). However, a large proportion of the ingested lactoferrin was not completely digested. Lactoferrin fragments containing the Lfcin region were analyzed by in situ hydrolysis with pepsin after being captured by the SEAC device. As much as 5.740 ± 0.702 x 10−5 mol/L of the partially degraded lactoferrin fragments were found to contain the Lfcin region, including peptide domains 17–43, 17–44, 12–44, 9–58, and 16–76 of bovine lactoferrin. These results show that bovine Lfcin can be produced in the human stomach after ingestion of an infant formula supplemented with bovine lactoferrin. It is now important to determine whether Lfcin is generated in the intestinal tract of formula-fed and breast-fed infants, and geriatric patients consuming foods enriched with lactoferrin.

A protective effect of bovine lactoferrin (Lf) during lethal bacteraemia has been reported in mice. Also, protective effects of orally administered bovine Lf have been reported in cases of intractable stomatitis in cats and Cryptocaryon irritans infection in red sea bream. In this study, we examined the effects of orally administered bovine Lf on the immune system of healthy volunteers.

Lactoferrin, an iron-binding glycoprotein of the transferrin family, is present in milk, saliva, mucous secretions and many other body fluids1. Lactoferrin is a major component of the secondary granules in neutrophils and this protein is released from activated neutrophils in the inflammatory response2. Many biological functions have been attributed to this protein, including roles in bacteriostasis in milk and at mucosal surfaces, immunomodulation, regulation of cell proliferation and intestinal iron transport3. Since the majority of the proposed functions of lactoferrin are based on the results of in vitro experiments, it seems clear that more in vivo studies are required for better understanding of the biological role of this protein. Considering the high similarities among the physicochemical properties of lactoferrins from several species with respect to molecular weight, primary structures4,5, three-dimensional structures6,7, and isoelectric point8, it seems possible that administered lactoferrin isolated from bovine milk may exert physiological activities in other animals and humans. In this paper, we outline research results demonstrating host defensive effects of orally administered bovine lactoferrin observed in several in vivo studies performed by our research group and some of our collaborators.

The human colon can be described as a complex microbial ecosystem, comprising several hundred bacterial species. Some of these enteric bacteria are beneficial to the host and have been shown to exert antimutagenic and anticarcinogenic properties. We have investigated the colon tumor inhibitory activity of Bifidobacterium longum, a lactic acid-producing enterobacterium. The modifying effects of this lactic culture on colonic mucosal and/or tumor cell proliferation, ODC activity and ras-p21 oncoprotein expression in colon carcinogenesis were also analyzed. Male F344 rats were fed a modified AIN-76A diet containing 0 or 2% lyophilized cultures of B. longum and s.c. administered azoxymethane (AOM) dissolved in normal saline at a dose of 15 mg/kg body wt, once weekly for 2 weeks. Vehicle controls received an equal volume of normal saline s.c. Animals were maintained on control or experimental diets until termination of the study. Animals intended for analysis of cell proliferation were killed 20 weeks after the second AOM injection, whereas animals intended for colon tumor analysis and measurement of ODC activity and ras-p21 expression were killed 40 weeks after the last AOM injection. The data demonstrate that dietary administration of lyophilized cultures of B. longum resulted in significant suppression of colon tumor incidence and tumor multiplicity and also reduced tumor volume. Results also revealed that ingestion of B. longum significantly inhibited AOM-induced cell proliferation, ODC activity and expression of ras-p21 oncoprotein. Data suggest that oral administration of probiotic B. longum exerts strong antitumor activity, as indicated by modulation of the intermediate biomarkers of colon cancer, and consequently reduced tumor outcome.

The antimicrobial role of ingested lactoferrin in the gut of infant animals remains unclear. To investigate the in vivo effects of lactoferrin on intestinal bacteria, we used milk-fed mice. Feeding bovine milk to mice resulted in a great increase in the number of intestinal Enterobacteriaceae in the gut. When mice were fed milk with bovine lactoferrin (bLf) added, the proliferation of Enterobacteriaceae was suppressed significantly. When milk-fed mice were orally inoculated with Clostridium ramosum C1, this strain proliferated in the gut and became the dominant component of the fecal microflora. In this model, administered bLf suppressed the proliferation of this strain and decreased the number of C. ramosum and other bacteria in the feces. A similar effect of bLf was observed after oral inoculation with other species of Clostridium. Compared with other proteins, only bLf showed these specific activities. Pepsin-hydrolyzed bLf (bLfH) showed the same effect on intestinal bacteria as undigested bLf. These results suggest that lactoferrin contained in mammalian milk may protect infant animals from gastrointestinal infections even after it has been digested to some extent.

The vitamin C activity of erythorbic acid (ErA) in ascorbic acid (AsA)-deficient guinea pigs was evaluated. The guinea pigs depleted AsA for 16 days were divided into two groups: one group (control group) was supplemented with 1, 5, or 100 mg/day AsA and the other group (experimental group) was supplemented with 1, 5, 20, or 100 mg/day ErA for 4 days. The contents of AsA and ErA in the tissues of guinea pigs were determined by using HPLC, and the activities of liver aniline hydroxylase, of serum alkaline phosphatase and the content of liver cytochrome P-450 were measured. The AsA tissue content of AsA-supplemented guinea pigs was much higher than the ErA tissue content of ErA-supplemented ones, and also, the activities of liver aniline hydroxy-lase, of serum alkaline phosphatase and the content of liver cytochrome P-450 of AsA-supplemented animals were much higher than those of ErA-supplemented animals, even when the supplemented amount of ErA was equal to that of AsA. Based on these results, the vitamin C activity of ErA seems to be much lower than that of AsA in the AsA-deficient guinea pigs. This suggested that the apparent vitamin C activity of ErA was dependent on the AsA tissue levels of guinea pigs.

In order to apply functionally active lactoferrin (Lf) to food products, the effect of pH on the heat stability of Lf was studied. Lf was easily denatured to an insoluble state by heat treatment under neutral or alkaline conditions, above pH 6. In contrast, it remained soluble after heat treatment under acidic conditions at pH 2 to 5, and the HPLC pattern of Lf heat-treated at pH 4 at 100°C for 5 min was the same as that of native Lf. Lf was found to be very thermostable at pH 4, and could be pasteurized or sterilized without any significant loss of its physicochemical properties. Lf was hydrolyzed by heat treatment at pH 2 to 3 at above 100°C, and its iron binding capacity and antigenicity were lost. But the antibacterial activity of the hydrolysate was found to be much stronger than that of native Lf. The antibacterial component of Lf hydrolysate produced by heat treatment at acidic pH was verified to be a peptide including the sequence of residues 1–54 from the N-terminal end of the bovine Lf molecule.